Monoclonal antibodies against ANGPTL3

ABSTRACT

Monoclonal antibodies that specifically bind to ANGPTL3 are provided. Monoclonal antibodies that neutralize at least one activity of ANGPTL3 are provided. Methods of treating a disorder of lipid metabolism using neutralizing monoclonal antibodies are provided.

This application claims the benefit of U.S. Provisional Application No.60/873,834, filed Dec. 8, 2006, which is incorporated by referenceherein for any purpose.

I. TECHNICAL FIELD

Monoclonal antibodies that specifically bind to angiopoietin-likeprotein 3 (ANGPTL3) are provided. Methods of using monoclonal antibodiesthat specifically bind to angiopoietin-like protein 3 (ANGPTL3) areprovided. Pharmaceutical compositions comprising monoclonal antibodiesthat specifically bind to angiopoietin-like protein 3 (ANGPTL3) areprovided.

II. INTRODUCTION

Angiopoeitin-like protein 3 (ANGPTL3) is conserved among severalmammalian species. Li, C. (2006) Curr. Opin. Lipidol. 17(2):152-156.ANGPTL3 contains an N-terminal coiled-coil domain and a C-terminalfibrinogen-like domain. Conklin, D. et al. (1999) Genomics 62:477-482.The N-terminal coiled-coil domain mediates oligomerization of ANGPTL3.Ge, H. et al. (2005) J. Lipid Res. 46(7):1484-1490. Oligomerized ANGPTL3undergoes proteolytic processing in vivo, resulting in the cleavage ofthe fibrinogen-like domain. Ono M. et al. (2003) J. Biol. Chem.278(43):41804-41809.

ANGPTL3 is expressed primarily in the liver. Koishi, R. et al. (2002)Nat. Genet. 30(2):151-157. ANGPTL3 appears to inhibit lipoprotein lipase(LPL) activity and decreases very low density lipoprotein (VLDL)clearance. Shimizugawa T. et al. (2002) J. Biol. Chem.277(37):33742-33748. The KK/San spontaneous mutant mouse contains aninsertion in exon 6 of the Angptl3 gene and shows notable serumhypolipidemia. Koishi et al. (2002) Nat. Genet., 30(2):151-157.Adenoviral-mediated expression of ANGPTL3 or direct administration ofANGPTL3 reversed the hypolipidemia in KK/San mice. Koishi et al. (2002)Nat. Genet. 30(2):151-157. An Angptl3 knockout mouse has been shown tohave reduced triglyceride levels in fed male mice and in fed or fastedfemale mice. Koster, A. et al., (2005) Endocrinol. 146:4943-4950. Thosemice have also been shown to have reduced cholesterol levels in both fedand fasted male and female mice. Koster, A. et al., (2005) Endocrinol.146:4943-4950. Angptl3 expression has been shown to be increased in bothstreptozotocin diabetic mice, in db/db mice, and in ob/ob mice. Inukai,K. et al., (2004) Biochem. Biophys. Res. Comm. 317(4):10751079;Shimamura, M. et al. (2004) Biochem. Biophys. Res. Comm.322(3):1080-1085.

III. SUMMARY

In certain embodiments, a monoclonal antibody that binds to ANGPTL3 andneutralizes at least one activity of ANGPTL3 is provided. In certainembodiments, the monoclonal antibody is a mouse monoclonal antibody. Incertain embodiments, the monoclonal antibody is a humanized monoclonalantibody. In certain embodiments, the monoclonal antibody is a humanmonoclonal antibody. In certain embodiments, the monoclonal antibodydecreases the level of at least one serum lipid in vivo.

In certain embodiments, the monoclonal antibody binds to an epitope ofANGPTL3 having the amino acid sequence of SEQ ID NO: 59. In certainembodiments, the monoclonal antibody binds to an epitope of ANGPTL3having the amino acid sequence of SEQ ID NO: 60. In certain embodiments,the monoclonal antibody binds to an epitope of ANGPTL3 having the aminoacid sequence of SEQ ID NO: 9. In certain embodiments, the monoclonalantibody binds to an epitope of ANGPTL3 having the amino acid sequenceof SEQ ID NO: 10.

In certain embodiments, the monoclonal antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 20 anda light chain variable region comprising the amino acid sequence of SEQID NO: 28. In certain embodiments, the monoclonal antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 22 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 30. In certain embodiments, the monoclonalantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 24 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 32. In certainembodiments, the monoclonal antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 64 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:68. In certain embodiments, the monoclonal antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:66 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 70.

In certain embodiments, the monoclonal antibody specifically binds tothe same epitope as antibody 4.7.1. In certain embodiments, themonoclonal antibody specifically binds to the same epitope as antibody4.8.3. In certain embodiments, the monoclonal antibody specificallybinds to the same epitope as antibody 4.9.1. In certain embodiments, themonoclonal antibody specifically binds to the same epitope as antibody1.315.1. In certain embodiments, the monoclonal antibody specificallybinds to the same epitope as antibody 5.35. In certain embodiments, themonoclonal antibody specifically binds to the same epitope as antibody5.50.

In certain embodiments, the monoclonal antibody is an antibody fragment.In certain embodiments, the monoclonal antibody is a scFv fragment. Incertain embodiments, the monoclonal antibody is a Fab fragment. Incertain embodiments, the monoclonal antibody is a F(ab′)₂ fragment. Incertain embodiments, the monoclonal antibody is a Fab′ fragment.

In certain embodiments, an isolated antibody that specifically binds toANGPTL3 is provided, comprising a heavy chain and a light chain, whereinthe heavy chain comprises: a) an amino acid sequence as set forth in anyone of SEQ ID NOs: 19 to 26 and 63 to 66; b) at least one amino acidsequence as set forth in SEQ ID NOs: 35, 36, and 37; c) at least oneamino acid sequence set forth in SEQ ID NOs: 38, 39, and 40; d) at leastone amino acid sequence as set forth in SEQ ID NOs: 41, 42, or 43; e) atleast one amino acid sequence as set forth in SEQ ID NOs: 53, 54, and55; f) at least one amino acid sequence as set forth in SEQ ID NOs: 71,72, and 73; or g) at least one amino acid sequence as set forth in SEQID NOs: 74, 75, and 76; wherein the antibody neutralizes at least oneactivity of ANGPTL3. In certain embodiments, the heavy chain of theantibody comprises a CDR1 as set forth in SEQ ID NO: 35, a CDR2 as setforth in SEQ ID NO: 36, and a CDR3 as set forth in SEQ ID NO: 37. Incertain embodiments, the heavy chain of the antibody comprises a CDR1 asset forth in SEQ ID NO: 38, a CDR2 as set forth in SEQ ID NO: 39, and aCDR3 as set forth in SEQ ID NO: 40. In certain embodiments, the heavychain of the antibody comprises a CDR1 as set forth in SEQ ID NO: 41, aCDR2 as set forth in SEQ ID NO: 42, and a CDR3 as set forth in SEQ IDNO: 43. In certain embodiments, the heavy chain of the antibodycomprises a CDR1 as set forth in SEQ ID NO: 53, a CDR2 as set forth inSEQ ID NO: 54, and a CDR3 as set forth in SEQ ID NO: 55. In certainembodiments, the heavy chain of the antibody comprises a CDR1 as setforth in SEQ ID NO: 71, a CDR2 as set forth in SEQ ID NO: 72, and a CDR3as set forth in SEQ ID NO: 73. In certain embodiments, the heavy chainof the antibody comprises a CDR1 as set forth in SEQ ID NO: 74, a CDR2as set forth in SEQ ID NO: 75, and a CDR3 as set forth in SEQ ID NO: 76.

In certain embodiments, an isolated antibody that specifically binds toANGPTL3 is provided, comprising a heavy chain and a light chain, whereinthe heavy chain comprises: a) an amino acid sequence as set forth in anyone of SEQ ID NOs: 19 to 26 and 63 to 66; b) at least one amino acidsequence as set forth in SEQ ID NOs: 35, 36, and 37; c) at least oneamino acid sequence set forth in SEQ ID NOs: 38, 39, and 40; d) at leastone amino acid sequence as set forth in SEQ ID NOs: 41, 42, or 43; e) atleast one amino acid sequence as set forth in SEQ ID NOs: 53, 54, and55; f) at least one amino acid sequence as set forth in SEQ ID NOs: 71,72, and 73; or g) at least one amino acid sequence as set forth in SEQID NOs: 74, 75, and 76; wherein the antibody neutralizes at least oneactivity of ANGPTL3; and wherein the light chain comprises: a) an aminoacid sequence as set forth in any one of SEQ ID NOs: 27 to 34 and 67 to70; b) at least one amino acid sequence as set forth in SEQ ID NOs: 44,45, and 46; c) at least one amino acid sequence set forth in SEQ ID NOs:47, 48, and 49; d) at least one amino acid sequence as set forth in SEQID NOs: 50, 51, or 52; e) at least one amino acid sequence as set forthin SEQ ID NOs: 56, 57, and 58; f) at least one amino acid sequence asset forth in SEQ ID NOs: 77, 78, and 79; or g) at least one amino acidsequence as set forth in SEQ ID NOs: 80, 81, and 82. In certainembodiments, the heavy chain of the antibody comprises a CDR1 as setforth in SEQ ID NO: 35, a CDR2 as set forth in SEQ ID NO: 36, and a CDR3as set forth in SEQ ID NO: 37; and the light chain of the antibodycomprises a CDR1 as set forth in SEQ ID NO: 44, a CDR2 as set forth inSEQ ID NO: 45, and a CDR3 as set forth in SEQ ID NO: 46. In certainembodiments, the heavy chain of the antibody comprises a CDR1 as setforth in SEQ ID NO: 38, a CDR2 as set forth in SEQ ID NO: 39, and a CDR3as set forth in SEQ ID NO: 40; and the light chain of the antibodycomprises a CDR1 as set forth in SEQ ID NO: 47, a CDR2 as set forth inSEQ ID NO: 48, and a CDR3 as set forth in SEQ ID NO: 49. In certainembodiments, the heavy chain of the antibody comprises a CDR1 as setforth in SEQ ID NO: 41, a CDR2 as set forth in SEQ ID NO: 42, and a CDR3as set forth in SEQ ID NO: 43; and the light chain of the antibodycomprises a CDR1 as set forth in SEQ ID NO: 50, a CDR2 as set forth inSEQ ID NO: 51, and a CDR3 as set forth in SEQ ID NO: 52. In certainembodiments, the heavy chain of the antibody comprises a CDR1 as setforth in SEQ ID NO: 53, a CDR2 as set forth in SEQ ID NO: 54, and a CDR3as set forth in SEQ ID NO: 55, and the light chain of the antibodycomprises a CDR1 as set forth in SEQ ID NO: 56, a CDR2 as set forth inSEQ ID NO: 57, and a CDR3 as set forth in SEQ ID NO: 58. In certainembodiments, the heavy chain of the antibody comprises a CDR1 as setforth in SEQ ID NO: 71, a CDR2 as set forth in SEQ ID NO: 72, and a CDR3as set forth in SEQ ID NO: 73, and the light chain of the antibodycomprises a CDR1 as set forth in SEQ ID NO: 77, a CDR2 as set forth inSEQ ID NO: 78, and a CDR3 as set forth in SEQ ID NO: 79. In certainembodiments, the heavy chain of the antibody comprises a CDR1 as setforth in SEQ ID NO: 74, a CDR2 as set forth in SEQ ID NO: 75, and a CDR3as set forth in SEQ ID NO: 76, and the light chain of the antibodycomprises a CDR1 as set forth in SEQ ID NO: 80, a CDR2 as set forth inSEQ ID NO: 81, and a CDR3 as set forth in SEQ ID NO: 82.

In certain embodiments, an isolated antibody that specifically binds toANGPTL3 is provided, comprising a heavy chain and a light chain, whereinthe light chain comprises: a) an amino acid sequence as set forth in anyone of SEQ ID NOs: 27 to 34 and 67 to 70; b) at least one amino acidsequence as set forth in SEQ ID NOs: 44, 45, and 46; c) at least oneamino acid sequence set forth in SEQ ID NOs: 47, 48, and 49; d) at leastone amino acid sequence as set forth in SEQ ID NOs: 50, 51, or 52; e) atleast one amino acid sequence as set forth in SEQ ID NOs: 56, 57, and58; f) at least one amino acid sequence as set forth in SEQ ID NOs: 77,78, and 79; or g) at least one amino acid sequence as set forth in SEQID NOs: 80, 81, and 82; wherein the antibody neutralizes at least oneactivity of ANGPTL3. In certain embodiments, the light chain of theantibody comprises a CDR1 as set forth in SEQ ID NO: 44, a CDR2 as setforth in SEQ ID NO: 45, and a CDR3 as set forth in SEQ ID NO: 46. Incertain embodiments, the light chain of the antibody comprises a CDR1 asset forth in SEQ ID NO: 47, a CDR2 as set forth in SEQ ID NO: 48, and aCDR3 as set forth in SEQ ID NO: 49. In certain embodiments, the lightchain comprises a CDR1 as set forth in SEQ ID NO: 50, a CDR2 as setforth in SEQ ID NO: 51, and a CDR3 as set forth in SEQ ID NO: 52. Incertain embodiments, the light chain comprises a CDR1 as set forth inSEQ ID NO: 56, a CDR2 as set forth in SEQ ID NO: 57, and a CDR3 as setforth in SEQ ID NO: 58. In certain embodiments, the light chaincomprises a CDR1 as set forth in SEQ ID NO: 77, a CDR2 as set forth inSEQ ID NO: 78, and a CDR3 as set forth in SEQ ID NO: 79. In certainembodiments, the light chain comprises a CDR1 as set forth in SEQ ID NO:80, a CDR2 as set forth in SEQ ID NO: 81, and a CDR3 as set forth in SEQID NO: 82.

In certain embodiments, a monoclonal antibody that bind to ANGPTL3 andneutralizes at least one activity of ANGPTL3 is provided, wherein theaffinity of the antibody for a peptide having an amino acid sequence ofSEQ ID NO: 9 is at least at least 3-fold greater than the affinity ofthe antibody for a peptide having an amino acid sequence of any one ofSEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO:88, SEQ ID NO: 90, and SEQ ID NO: 92. In certain embodiments, theaffinity of the antibody for a peptide having an amino acid sequence ofSEQ ID NO: 9 is at least at least 3-fold greater than the affinity ofthe antibody for each of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87,SEQ ID NO: 88, and SEQ ID NO: 90. In certain embodiments, a monoclonalantibody that bind to ANGPTL3 and neutralizes at least one activity ofANGPTL3 is provided, wherein the antibody binds to a peptide having theamino acid sequence of SEQ ID NO: 9 with a K_(D) of less than 50 nM. Incertain embodiments, the antibody binds to a peptide having the aminoacid sequence of SEQ ID NO: 9 with a K_(D) of less than 30 nM. Incertain embodiments, the antibody binds to a peptide having the aminoacid sequence of SEQ ID NO: 9 with a K_(D) of less than 10 nM. Incertain embodiments, the antibody binds to a peptide having the aminoacid sequence of SEQ ID NO: 9 with a K_(D) of less than 5 nM.

In certain embodiments, a pharmaceutical composition is providedcomprising an antibody as described above. In certain embodiments, amethod of treating a disorder of lipid metabolism is provided, whereinthe method comprises administering to a patient an effective amount ofthe pharmaceutical composition. In certain embodiments, a method ofdecreasing the level of one or more serum lipids is provided, whereinthe method comprises administering to a patient an effective amount ofthe pharmaceutical composition. In certain embodiments, a method oftreating hypertriglyceridemia is provided, wherein the method comprisesadministering to a patient an effective amount of the pharmaceuticalcomposition. In certain embodiments, a method of treatinghypercholesterolemia is provided, wherein the method comprisesadministering to a patient an effective amount of the pharmaceuticalcomposition. In certain embodiments, a method of treating obesity isprovided, wherein the method comprises administering to a patient aneffective amount of the pharmaceutical composition. In certainembodiments, a method of treating diabetes is provided, wherein themethod comprises administering to a patient an effective amount of thepharmaceutical composition. In certain embodiments, a method of treatingischaemic heart disease is provided, wherein the method comprisesadministering to a patient an effective amount of the pharmaceuticalcomposition. In certain embodiments, a method of treating metabolicsyndrome is provided, wherein the method comprises administering to apatient an effective amount of the pharmaceutical composition.

IV. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows fed and fasted serum triglyceride levels in mice 4 daysafter injection with antibodies 1.315.1, 4.7.1, 4.8.3, 4.9.1, andcontrol antibody anti-KLH, as described in Example J.

FIG. 2 shows fed and fasted serum cholesterol levels in mice 4 daysafter injection with antibodies 1.315.1, 4.7.1, 4.8.3, 4.9.1, andcontrol antibody anti-KLH, as described in Example J.

FIG. 3 shows fed and fasted serum triglyceride levels in mice 4 daysafter injection with anti-ANGPTL3 antibody 4.8.3, anti-ANGPTL4 antibody14D12, and control antibody anti-KLH, as described in Example J.

FIG. 4 shows fed and fasted serum cholesterol levels in mice 4 daysafter injection with anti-ANGPTL3 antibody 4.8.3, anti-ANGPTL4 antibody14D12, and control antibody anti-KLH, as described in Example J.

FIG. 5 shows fed and fasted serum triglyceride levels in mice 4 daysafter injection with anti-ANGPTL3 antibodies 4.7.1, 4.8.3, 4.9.1,anti-ANGPTL4 antibody 14D12, and control antibody anti-KLH, as describedin Example J.

FIG. 6 shows fed and fasted serum cholesterol levels in mice 4 daysafter injection with anti-ANGPTL3 antibodies 4.7.1, 4.8.3, 4.9.1,anti-ANGPTL4 antibody 14D12, and control antibody anti-KLH, as describedin Example J.

FIG. 7 shows serum triglyceride levels in mice 4 days (top panel) and 8days (bottom panel) after injection with antibodies 1.125.1, 1.132.1,1.173.2, 1.315.1, 1.424.1, 1.431.1, and control antibody anti-KLH, asdescribed in Example J.

FIG. 8 shows serum triglyceride levels in mice after injection withvarious dosages of Ad5-hAngptl3T virus or 2×10⁹ vp of control virus, asdescribed in Example B.

FIG. 9 shows binding of antibodies 4.1.1, 4.4.1, 4.6.3, 4.7.1, 4.8.1,4.8.2, 4.8.3, and 4.9.1 to ANGPTL3 SP1, ANGPTL3T, and control proteinBTS324, as described in Example H.

FIG. 10 shows an alignment of the heavy chain variable regions ofantibodies 4.7.1 (SEQ ID NO: 19), 4.8.3 (SEQ ID NO: 21), and 4.9.1 (SEQID NO: 23). The heavy chain consensus sequence is also shown (SEQ ID NO:25), as well as the regions of the heavy chain sequences correspondingto the signal peptide (SP), framework region 1 (FWR1),complementarity-determining region 1 (CDR1), framework region 2 (FWR2),complementarity-determining region 2 (CDR2), framework region 3 (FWR3),complementarity-determining region 3 (CDR3), and framework region 4(FWR4).

FIG. 11 shows an alignment of the light chain variable regions ofantibodies 4.7.1 (SEQ ID NO: 27), 4.8.3 (SEQ ID NO: 29), and 4.9.1 (SEQID NO: 31). The light chain consensus sequence is also shown (SEQ ID NO:33), as well as the regions of the light chain sequences correspondingto the signal peptide (SP), framework region 1 (FWR1),complementarity-determining region 1 (CDR1), framework region 2 (FWR2),complementarity-determining region 2 (CDR2), framework region 3 (FWR3),complementarity-determining region 3 (CDR3), and framework region 4(FWR4).

FIG. 12 shows LPL activity in vitro after treatment with mousemonoclonal anti-ANGPTL3 antibodies 4.9.1, 4.8.3, 1.315.1, 4.7.1, and1.173.2, as described in Example L.

FIG. 13 shows the in vivo pharmacokinetics of certain mouse monoclonalanti-ANGPTL3 antibodies, as described in Example N.

FIG. 14 shows binding of antibodies 4.7.1, 4.8.3, 4.9.1, 5.35, and 5.50to the alanine-scanning mutants of the SP1 peptide, as described inExample H.

FIG. 15 shows serum triglyceride levels in mice 4 days (top panel) and 7days (bottom panel) after injection with antibody 5.35 or 5.50, asdescribed in Example J.

FIG. 16 shows serum cholesterol levels in mice 4 days (top panel) and 7days (bottom panel) after injection with antibody 5.35 or 5.50, asdescribed in Example J.

FIG. 17 shows the in vivo pharmacokinetics of antibodies 5.35 and 5.50,as described in Example N.

FIG. 18 shows the in vivo pharmacokinetics of antibodies 4.7.1, 4.9.1,5.35, and 5.50, as described in Example N.

FIG. 19 shows the reduction in serum triglycerides and serum cholesterolin ApoE mice injected with antibodies 5.35 and 5.50 and control antibodyanti-KLH.

V. DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the word “a” or “an”means “at least one” unless specifically stated otherwise. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including,” as well as other forms,such as “includes” and “included,” is not limiting. Also, terms such as“element” or “component” encompass both elements or componentscomprising one unit and elements or components that comprise more thanone unit unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises are hereby expressly incorporated by reference intheir entirety for any purpose. In the event that one or more of theincorporated literature and similar materials defines a term thatcontradicts that term's definition in this application, this applicationcontrols.

A. Certain Definitions

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers containing naturally occurring aminoacids as well as amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid. The amino acid polymers can be of any length.

The term “antibody,” as used herein, refers to an intact antibody or afragment of an antibody that competes with the intact antibody forantigen binding. Antibody fragments include, but are not limited to,Fab, Fab′, F(ab′)₂, Fv, scFv, Fd, diabodies, and other antibodyfragments that retain at least a portion of the variable region of anintact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134. Incertain embodiments, antibody fragments are produced by enzymatic orchemical cleavage of intact antibodies. In certain embodiments, antibodyfragments are produced by recombinant DNA techniques.

The term “native polypeptide” refers to a naturally occurringpolypeptide. The term “native antibody” refers to a naturally occurringantibody.

The term “monoclonal antibody” refers to an antibody from asubstantially homogeneous population of antibodies that specificallybind to the same epitope. In certain embodiments, a monoclonal antibodyis secreted by a hybridoma. In certain such embodiments, a hybridoma isproduced according to certain methods known to those skilled in the art.See, e.g., Kohler and Milstein (1975) Nature 256: 495-499. In certainembodiments, a monoclonal antibody is produced using recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). In certain embodiments, amonoclonal antibody refers to an antibody fragment isolated from a phagedisplay library. See, e.g., Clackson et al. (1991) Nature 352: 624-628,and Marks et al. (1991) J. Mol. Biol. 222: 581-597. For various othermonoclonal antibody production techniques, see, e.g., Harlow and Lane(1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

A “chimeric” antibody refers to an antibody made up of components fromat least two different sources. In certain embodiments, a chimericantibody comprises a portion of an antibody derived from a first speciesfused to another molecule, e.g., a portion of an antibody derived from asecond species. In certain such embodiments, a chimeric antibodycomprises a portion of an antibody derived from a non-human animal fusedto a portion of an antibody derived from a human. In certain suchembodiments, a chimeric antibody comprises all or a portion of avariable region of an antibody derived from a non-human animal fused toa constant region of an antibody derived from a human.

A “humanized” antibody refers to a non-human antibody that has beenmodified so that it more closely matches (in amino acid sequence) ahuman antibody. A humanized antibody is thus a type of chimericantibody. In certain embodiments, amino acid residues outside of theantigen binding residues of the variable region of the non-humanantibody are modified. In certain embodiments, a humanized antibody isconstructed by replacing all or a portion of a complementaritydetermining region (CDR) of a human antibody with all or a portion of aCDR from another antibody, such as a non-human antibody, having thedesired antigen binding specificity. In certain embodiments, a humanizedantibody comprises variable regions in which all or substantially all ofthe CDRs correspond to CDRs of a non-human antibody and all orsubstantially all of the framework regions (FRs) correspond to FRs of ahuman antibody. In certain such embodiments, a humanized antibodyfurther comprises a constant region (Fc) of a human antibody.

The term “human antibody” refers to a monoclonal antibody that containshuman antibody sequences and does not contain antibody sequences from anon-human animal. In certain embodiments, a human antibody may containsynthetic sequences not found in native antibodies. The term is notlimited by the manner in which the antibodies are made. For example, invarious embodiments, a human antibody may be made in a transgenic mouse,by phage display, by human B-lymphocytes, or by recombinant methods.

The term “neutralizing antibody” or “antibody that neutralizes” refersto an antibody that reduces at least one activity of a polypeptidecomprising the epitope to which the antibody specifically binds. Incertain embodiments, a neutralizing antibody reduces an activity invitro and/or in vivo.

The term “antigen-binding site” refers to a portion of an antibodycapable of specifically binding an antigen. In certain embodiments, anantigen-binding site is provided by one or more antibody variableregions.

The term “epitope” refers to any polypeptide determinant capable ofspecifically binding to an immunoglobulin or a T-cell receptor. Incertain embodiments, an epitope is a region of an antigen that isspecifically bound by an antibody. In certain embodiments, an epitopemay include chemically active surface groupings of molecules such asamino acids, sugar side chains, phosphoryl, or sulfonyl groups. Incertain embodiments, an epitope may have specific three dimensionalstructural characteristics (e.g., a “conformational” epitope) and/orspecific charge characteristics.

An epitope is defined as “the same” as another epitope if a particularantibody specifically binds to both epitopes. In certain embodiments,polypeptides having different primary amino acid sequences may compriseepitopes that are the same. In certain embodiments, epitopes that arethe same may have different primary amino acid sequences. Differentantibodies are said to bind to the same epitope if they compete forspecific binding to that epitope.

An antibody “specifically binds” an antigen when it preferentiallyrecognizes the antigen in a complex mixture of proteins and/ormacromolecules. In certain embodiments, an antibody comprises anantigen-binding site that specifically binds to a particular epitope. Incertain such embodiments, the antibody is capable of binding differentantigens so long as the different antigens comprise that particularepitope. In certain instances, for example, homologous proteins fromdifferent species may comprise the same epitope. In certain embodiments,an antibody is said to specifically bind an antigen when thedissociation constant (K_(D)) is ≦1 μM, in certain embodiments, when thedissociation constant is ≦100 nM, and in certain embodiments, when thedissociation constant is ≦10 nM.

The term “ANGPTL3” refers to an angiopoietin like protein 3 having anamino acid sequence from any vertebrate or mammalian source, including,but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine,ovine, primate, monkey, and guinea pig, unless specified otherwise. Theterm also refers to fragments and variants of native ANGPTL3 thatmaintain at least one in vivo or in vitro activity of a native ANGPTL3.The term encompasses full-length unprocessed precursor forms of ANGPTL3as well as mature forms resulting from post-translational cleavage ofthe signal peptide and forms resulting from proteolytic processing ofthe fibrinogen domain. In certain embodiments, a full-length,unprocessed mouse ANGPTL3 has the amino acid sequence set forth in SEQID NO: 1 (Genbank accession no. NP_(—)038941). In certain embodiments, afull-length, unprocessed human ANGPTL3 has the amino acid sequence setforth in SEQ ID NO: 3 (Genbank accession no. NP_(—)055310).

The term “Angptl3” refers to a nucleic acid encoding ANGPTL3.

The term “LPL” refers to a lipoprotein lipase having an amino acidsequence from any vertebrate or mammalian source, including, but notlimited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine,primate, monkey, and guinea pig. In certain embodiments, a lipoproteinlipase catalyzes the hydrolysis of triacylglycerol in chylomicrons andvery low density lipoproteins (VLDLs) into diacylglycerol and a freefatty acid anion. In certain embodiments, a lipoprotein lipase is alsoable to hydrolyze diacylglycerol.

The term “agent” refers to a chemical compound, a mixture of chemicalcompounds, a biological macromolecule, or an extract made frombiological materials.

The term “antagonist of ANGPTL3” refers to an agent that reduces anactivity of ANGPTL3.

The term “agonist of ANGPTL3” refers to an agent that increases anactivity of ANGPTL3.

The term “patient” includes human and animal subjects. In certainembodiments, a patient is a mammal. In certain such embodiments, apatient is a human.

A “fragment” of a reference polypeptide refers to a contiguous stretchof amino acids from any portion of the reference polypeptide. A fragmentmay be of any length that is less than the length of the referencepolypeptide.

A “variant” of a reference polypeptide refers to a polypeptide havingone or more amino acid substitutions, deletions, or insertions relativeto the reference polypeptide.

A “conservative” amino acid substitution refers to the substitution ofan amino acid in a polypeptide with another amino acid having similarproperties, such as size or charge. In certain embodiments, apolypeptide comprising a conservative amino acid substitution maintainsat least one activity of the unsubstituted polypeptide. A conservativeamino acid substitution may encompass non-naturally occurring amino acidresidues, which are typically incorporated by chemical peptide synthesisrather than by synthesis in biological systems. These include, but arenot limited to, peptidomimetics and other reversed or inverted forms ofamino acid moieties.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: H is, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of a human antibodythat are homologous with non-human antibodies, or into thenon-homologous regions of the molecule.

In making substitutions, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein, in certain instances, isunderstood in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982).It is known that in certain instances, certain amino acids may besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, in certain embodiments, thesubstitution of amino acids whose hydropathic indices are within ±2 isincluded. In certain embodiments, those which are within ±1 areincluded, and in certain embodiments, those within ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions Original Residue ExemplarySubstitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu CysSer, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu,Val, Met, Ala, Phe, Norleucine Leu Norleucine, Ile, Val, Met, Ala, PheLys Arg, 1,4 Diamino-butyric Acid, Gln, Asn Met Leu, Phe, Ile Phe Leu,Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe TyrTrp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala, Norleucine

A skilled artisan will be able to determine suitable variants of apolypeptide as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In certainembodiments, one can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In certain embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, in certain embodiments, one skilled in the art can reviewstructure-function studies identifying residues in similar polypeptidesthat are important for activity or structure. In view of such acomparison, in certain embodiments, one can predict the importance ofamino acid residues in a protein that correspond to amino acid residueswhich are important for activity or structure in similar proteins. Incertain embodiments, one skilled in the art may opt for chemicallysimilar amino acid substitutions for such predicted important amino acidresidues.

In certain embodiments, one skilled in the art can also analyze thethree-dimensional structure and amino acid sequence in relation to thatstructure in similar polypeptides. In certain embodiments, in view ofsuch information, one skilled in the art may predict the alignment ofamino acid residues of an antibody with respect to its three dimensionalstructure. In certain embodiments, one skilled in the art may choose notto make radical changes to amino acid residues predicted to be on thesurface of the protein, since such residues may be involved in importantinteractions with other molecules. Moreover, in certain embodiments, oneskilled in the art may generate test variants containing a single aminoacid substitution at each desired amino acid residue. In certainembodiments, the variants can then be screened using activity assaysknown to those skilled in the art. In certain embodiments, such variantscould be used to gather information about suitable variants. Forexample, in certain embodiments, if one discovered that a change to aparticular amino acid residue resulted in destroyed, undesirablyreduced, or unsuitable activity, variants with such a change may beavoided. In other words, in certain embodiments, based on informationgathered from such routine experiments, one skilled in the art canreadily determine the amino acids where further substitutions should beavoided either alone or in combination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See, e.g., Moult J., Curr. Opin. Biotechnol.(1996) 7(4):422-427; Chou et al., (1974) Biochemistry, 13(2):222-245;Chou et al. (1974) Biochemistry 113(2):211-222; Chou et al. (1978); Adv.Enzymol. Relat. Areas Mol. Biol. 47:45-148; Chou et al. (1976) Ann. Rev.Biochem. 47:251-276; and Chou et al. (1979) Biophys. J. 26:367-384.Moreover, computer programs are currently available to assist withpredicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The growth of the protein structural database (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's structure. See, e.g., Holm et al.(1999) Nucl. Acid. Res. 27(1):244-247. It has been suggested (Brenner etal., (1997) Curr. Op. Struct. Biol. 7(3):369-376) that there are alimited number of folds in a given polypeptide or protein and that oncea critical number of structures have been resolved, structuralprediction will become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(see, e.g., Jones, D. (1:997) Curr. Opin. Struct. Biol. 7(3):377-387;Sippl et al. (1996) Structure 4(1):15-19), “profile analysis” (see,e.g., Bowie et al., (1991) Science 253:164-170; Gribskov et al., (1990)Meth. Enzym. 183:146-159; Gribskov et al. (1987) Proc. Nat. Acad. Sci.USA 84(13):4355-4358), and “evolutionary linkage” (see, e.g., Holm etal. (1999) Nucl. Acid. Res. 27(1):244-247; and Brenner et al. (1997)Curr. Op. Struct. Biol. 7(3):369-376 (1997)).

In certain embodiments, a variant of a reference antibody includes aglycosylation variant wherein the number and/or type of glycosylationsites have been altered relative to the amino acid sequence of thereference antibody. In certain embodiments, a variant of a polypeptidecomprises a greater or a lesser number of N-linked glycosylation sitesrelative to a native polypeptide. An N-linked glycosylation site ischaracterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the aminoacid residue designated as X may be any amino acid residue exceptproline. The substitution of amino acid residues to create this sequenceprovides a potential new site for the addition of an N-linkedcarbohydrate chain. Alternatively, substitutions which eliminate thissequence will remove an existing N-linked carbohydrate chain. In certainembodiments, a rearrangement of N-linked carbohydrate chains isprovided, wherein one or more N-linked glycosylation sites (typicallythose that are naturally occurring) are eliminated and one or more newN-linked sites are created. Exemplary antibody variants include cysteinevariants wherein one or more cysteine residues are deleted from orsubstituted for another amino acid (e.g., serine) relative to the aminoacid sequence of the reference antibody. In certain embodiments,cysteine variants may be useful when antibodies must be refolded into abiologically active conformation such as after the isolation ofinsoluble inclusion bodies. In certain embodiments, cysteine variantshave fewer cysteine residues than the native polypeptide. In certainembodiments, cysteine variants have an even number of cysteine residuesto minimize interactions resulting from unpaired cysteines.

According to certain embodiments, amino acid substitutions are thosewhich: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (4) confer ormodify other physiochemical or functional properties on suchpolypeptides. According to certain embodiments, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) may be made in a naturally-occurring sequence (in certainembodiments, in the portion of the polypeptide outside the domain(s)forming intermolecular contacts). In certain embodiments, a conservativeamino acid substitution typically may not substantially change thestructural characteristics of the reference sequence (e.g., in certainembodiments, a replacement amino acid should not tend to break a helixthat occurs in the reference sequence, or disrupt other types ofsecondary structure that characterizes the reference sequence). Examplesof certain art-recognized polypeptide secondary and tertiary structuresare described, for example, in Proteins, Structures and MolecularPrinciples (Creighton, Ed., W. H. Freeman and Company, New York (1984));Introduction to Protein Structure (C. Branden and J. Tooze, eds.,Garland Publishing, New York, N.Y. (1991)); and Thornton, J. M. et al.(1991) Nature 354:105-106.

“Percent identity” or “% identity,” with reference to nucleic acidsequences, refers to the percentage of identical nucleotides between atleast two polynucleotide sequences aligned using the Basic LocalAlignment Search Tool (BLAST) engine. See Tatusova et al. (1999) FEMSMicrobiol. Lett. 174:247-250. The BLAST engine (version 2.2.10) isprovided to the public by the National Center for BiotechnologyInformation (NCBI), Bethesda, Md. To align two polynucleotide sequences,the “Blast 2 Sequences” tool is used, which employs the “blastn” programwith parameters set at default values as follows:

Matrix: not applicable

Reward for match: 1

Penalty for mismatch: −2

Open gap: 5 penalties

Extension gap: 2 penalties

Gap_x dropoff: 50

Expect: 10.0

Word size: 11

Filter: on

“Percent identity” or “% identity,” with reference to polypeptidesequences, refers to the percentage of identical amino acids between atleast two polypeptide sequences aligned using the Basic Local AlignmentSearch Tool (BLAST) engine. See Tatusova et al. (1999) FEMS Microbiol.Lett. 174:247-250. The BLAST engine (version 2.2.10) is provided to thepublic by the National Center for Biotechnology Information (NCBI),Bethesda, Md. To align two polypeptide sequences, the “Blast 2Sequences” tool is used, which employs the “blastp” program withparameters set at default values as follows:

Matrix: BLOSUM62

Open gap: 11 penalties

Extension gap: 1 penalty

Gap_x dropoff: 50

Expect: 10.0

Word size: 3

Filter: on

The term “effective dose” or “effective amount” refers to an amount ofan agent, e.g., a neutralizing antibody, that results in the reductionof symptoms in a patient or results in a desired biological outcome. Incertain embodiments, an effective dose or effective amount is sufficientto reduce at least one activity of ANGPTL3. In certain embodiments, aneffective dose or effective amount is determined as described below,Part V.G.

The term “treatment” encompasses both therapeutic andprophylactic/preventative measures unless otherwise indicated. Those inneed of treatment include, but are not limited to, individuals alreadyhaving a particular condition or disorder as well as individuals who areat risk of acquiring a particular condition or disorder (e.g., thoseneeding prophylactic/preventative measures). The term “treating” refersto administering an agent to a patient for therapeutic and/orprophylactic/preventative purposes.

A “therapeutic agent” refers to an agent that may be administered invivo to bring about a therapeutic and/or prophylactic/preventativeeffect.

A “therapeutic antibody” refers to an antibody that may be administeredin vivo to bring about a therapeutic and/or prophylactic/preventativeeffect.

The terms “isolated nucleic acid” and “isolated polynucleotide” are usedinterchangeably. Exemplary isolated polynucleotides include, but are notlimited to, genomic DNA, RNA, cDNA, synthetic DNA or RNA or somecombination thereof. An “isolated polynucleotide” (1) is not associatedwith all or a portion of a polynucleotide in which the “isolatedpolynucleotide” is found in nature, (2) is linked to a polynucleotide towhich it is not linked in nature, or (3) does not occur in nature aspart of a larger sequence.

B. Structure of Native Antibodies and Certain Antibody Fragments

A native antibody typically has a tetrameric structure. A tetramertypically comprises two identical pairs of polypeptide chains, each pairhaving one light chain (in certain embodiments, about 25 kDa) and oneheavy chain (in certain embodiments, about 50-70 kDa). In a nativeantibody, a heavy chain comprises a variable region, V_(H), and threeconstant regions, C_(H)1, C_(H)2, and C_(H)3. The V_(H) domain is at theamino-terminus of the heavy chain, and the C_(H)3 domain is at thecarboxy-terminus. In a native antibody, a light chain comprises avariable region, V_(L), and a constant region, C_(L). The variableregion of the light chain is at the amino-terminus of the light chain.In a native antibody, the variable regions of each light/heavy chainpair typically form the antigen binding site. The constant regions aretypically responsible for effector function.

Native human light chains are typically classified as kappa and lambdalight chains. Native human heavy chains are typically classified as mu,delta, gamma, alpha, or epsilon, and define the antibody's isotype asIgM, IgD, IgG, IgA, and IgE, respectively. IgG has subclasses,including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM hassubclasses including, but not limited to, IgM1 and IgM2. IgA hassubclasses including, but not limited to, IgA1 and IgA2. Within nativehuman light and heavy chains, the variable and constant regions aretypically joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 10 more aminoacids. See, e.g., Fundamental Immunology (1989) Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y.).

In a native antibody, the variable regions typically exhibit the samegeneral structure in which relatively conserved framework regions (FRs)are joined by three hypervariable regions, also called complementaritydetermining regions (CDRs). The CDRs from the two chains of each pairtypically are aligned by the framework regions, which may enable bindingto a specific epitope. From N-terminus to C-terminus, both light andheavy chain variable regions typically comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The CDRs on the heavy chain are referredto as H1, H2, and H3, while the CDRs on the light chain are referred toas L1, L2, and L3. Typically, CDR3 is the greatest source of moleculardiversity within the antigen-binding site. H3, for example, in certaininstances, can be as short as two amino acid residues or greater than26. The assignment of amino acids to each domain is typically inaccordance with the definitions of Kabat et al. (1991) Sequences ofProteins of Immunological Interest (National Institutes of Health,Publication No. 91-3242, vols. 1-3, Bethesda, Md.); Chothia, C., andLesk, A. M. (1987) J. Mol. Biol. 196:901-917; or Chothia, C. et al.Nature 342:878-883 (1989). In the present application, the term “CDR”refers to a CDR from either the light or heavy chain, unless otherwisespecified.

A “Fab” fragment comprises one light chain and the C_(H)1 and variableregion of one heavy chain. The heavy chain of a Fab molecule cannot forma disulfide bond with another heavy chain molecule. A “Fab′” fragmentcomprises one light chain and one heavy chain that comprises additionalconstant region, extending between the C_(H)1 and C_(H)2 domains. Aninterchain disulfide bond can be formed between two heavy chains of aFab′ fragment to form a “F(ab′)₂” molecule.

An “Fv” fragment comprises the variable regions from both the heavy andlight chains, but lacks the constant regions. A single-chain Fv (scFv)fragment comprises heavy and light chain variable regions connected by aflexible linker to form a single polypeptide chain with anantigen-binding region. Exemplary single chain antibodies are discussedin detail in WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. Incertain instances, a single variable region (i.e., a heavy chainvariable region or a light chain variable region) may have the abilityto recognize and bind antigen.

As used herein, the term “heavy chain” refers to a polypeptidecomprising sufficient heavy chain variable region sequence to conferantigen specificity either alone or in combination with a light chain.

As used herein, the term “light chain” refers to a polypeptidecomprising sufficient light chain variable region sequence to conferantigen specificity either alone or in combination with a heavy chain.

C. Certain Antibodies

In certain embodiments, monoclonal antibodies that specifically bind toANGPTL3 are provided. In certain such embodiments, the monoclonalantibodies are neutralizing monoclonal antibodies that reduce at leastone activity of ANGPTL3 in vivo and/or in vitro.

In certain embodiments, a neutralizing monoclonal antibody againstANGPTL3 reduces at least one serum lipid level in vivo. In certainembodiments, a neutralizing monoclonal antibody against ANGPTL3 reducesserum triglyceride levels in vivo. In certain embodiments, aneutralizing monoclonal antibody reduces total cholesterol levels invivo. In certain embodiments, a neutralizing monoclonal antibody againstANGPTL3 reduces free fatty acid (FFA) levels in vivo. In certainembodiments, a neutralizing monoclonal antibody against ANGPTL3 reducesat least two of those levels in vivo. In certain embodiments, aneutralizing monoclonal antibody against ANGPTL3 reduces at least threeof those levels in vivo.

In certain embodiments, a neutralizing monoclonal antibody againstANGPTL3 reduces serum triglycerides in LDLr knockout mice in vivo. Incertain embodiments, a neutralizing monoclonal antibody against ANGPTL3reduces total cholesterol in LDLr knockout mice in vivo. In certainembodiments, a neutralizing monoclonal antibody against ANGPTL3 reducesserum triglycerides in ApoE knockout mice in vivo. In certainembodiments, a neutralizing monoclonal antibody against ANGPTL3 reducestotal cholesterol in ApoE knockout mice in vivo. In certain embodiments,a neutralizing monoclonal antibody against ANGPTL3 reduces serumtriglycerides in db/db mice in vivo. In certain embodiments, aneutralizing monoclonal antibody against ANGPTL3 reduces totalcholesterol in db/db mice in vivo.

In certain embodiments, neutralizing monoclonal antibodies thatspecifically bind to mouse ANGPTL3 are provided. In certain embodiments,neutralizing monoclonal antibodies that specifically bind to humanANGPTL3 are provided. In certain embodiments, neutralizing monoclonalantibodies that specifically bind to the same epitope in ANGPTL3 fromdifferent species (i.e., antibodies that demonstrate cross-reactivity)are provided. In certain such embodiments, the antibodies specificallybind to both mouse ANGPTL3 and human ANGPTL3.

In certain embodiments, neutralizing monoclonal antibodies thatspecifically bind to an epitope within the N-terminal coiled-coil domainof ANGPTL3 are provided. In certain embodiments, neutralizing monoclonalantibodies that specifically bind to an epitope within the N-terminalcoiled-coil domain of mouse ANGPTL3 are provided. In certainembodiments, neutralizing monoclonal antibodies specifically bind to anepitope within a region of mouse ANGPTL3 from residue 17 to residue 240of SEQ ID NO: 1 (SEQ ID NO: 59). In certain embodiments, neutralizingmonoclonal antibodies specifically bind to the SP1 region of mouseANGPTL3 from residue 32 to residue 57 of SEQ ID NO: 1 (SEQ ID NO: 9). Incertain embodiments, neutralizing monoclonal antibodies specificallybind to an epitope within the SP1 region of mouse ANGPTL3 that includesamino acids 34 to 37 and 40 of SEQ ID NO: 1 (amino acids 3 to 6 and 9 ofSEQ ID NO: 9). In certain embodiments, neutralizing monoclonalantibodies specifically bind to an epitope within the SP1 region ofmouse ANGPTL3 that includes amino acids 33 to 37 and 40 of SEQ ID NO: 1(amino acids 2 to 6 and 9 of SEQ ID NO: 9). In certain embodiments,neutralizing monoclonal antibodies specifically bind to an epitopewithin the SP1 region of mouse ANGPTL3 that includes amino acids 34 to37, 40, and 42 of SEQ ID NO: 1 (amino acids 3 to 6, 9, and 11 of SEQ IDNO: 9). In certain embodiments, neutralizing monoclonal antibodiesspecifically bind to an epitope within the SP1 region of mouse ANGPTL3that includes amino acids 34 to 37 and 40 to 42 of SEQ ID NO: 1 (aminoacids 3 to 6 and 9 to 11 of SEQ ID NO: 9).

In certain embodiments, neutralizing monoclonal antibodies thatspecifically bind to an epitope within the N-terminal coiled-coil domainof human ANGPTL3 are provided. In certain embodiments, neutralizingmonoclonal antibodies specifically bind to an epitope within a region ofhuman ANGPTL3 from residue 20 to residue 143 of SEQ ID NO: 3 (SEQ ID NO:60). In certain embodiments, neutralizing monoclonal antibodiesspecifically bind to the SP1 region of human ANGPTL3 from residue 32 toresidue 57 of SEQ ID NO: 3 (SEQ ID NO: 10). In certain embodiments,neutralizing monoclonal antibodies specifically bind to an epitopewithin the SP1 region of mouse ANGPTL3 that includes amino acids 34 to37 and 40 of SEQ ID NO: 3 (amino acids 3 to 6 and 9 of SEQ ID NO: 10).In certain embodiments, neutralizing monoclonal antibodies specificallybind to an epitope within the SP1 region of mouse ANGPTL3 that includesamino acids 33 to 37 and 40 of SEQ ID NO: 3 (amino acids 2 to 6 and 9 ofSEQ ID NO: 10). In certain embodiments, neutralizing monoclonalantibodies specifically bind to an epitope within the SP1 region ofmouse ANGPTL3 that includes amino acids 34 to 37, 40, and 42 of SEQ IDNO: 3 (amino acids 3 to 6, 9, and 11 of SEQ ID NO: 10). In certainembodiments, neutralizing monoclonal antibodies specifically bind to anepitope within the SP1 region of mouse ANGPTL3 that includes amino acids34 to 37 and 40 to 42 of SEQ ID NO: 3 (amino acids 3 to 6 and 9 to 11 ofSEQ ID NO: 10).

In various embodiments, a neutralizing monoclonal antibody binds to apeptide having an amino acid sequence of SEQ ID NO: 9 with at least2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least7-fold, or at least 10-fold greater affinity than the neutralizingmonoclonal antibody binds to a peptide having an amino acid sequence ofany one of SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87,SEQ ID NO: 88, SEQ ID NO: 90, and SEQ ID NO: 92. In various embodiments,a neutralizing monoclonal antibody binds to a peptide having an aminoacid sequence of SEQ ID NO: 9 with at least 2-fold, at least 3-fold, atleast 4-fold, at least 5-fold, at least 7-fold, or at least 10-foldgreater affinity than the neutralizing monoclonal antibody binds to eachof SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ IDNO: 88, SEQ ID NO: 90, and SEQ ID NO: 92. In certain embodiments,affinity is determined as described in Example H (2).

In certain embodiments, neutralizing monoclonal antibodies are non-humanmonoclonal antibodies. In certain such embodiments, neutralizingmonoclonal antibodies are rodent monoclonal antibodies. In certain suchembodiments, neutralizing monoclonal antibodies are mouse monoclonalantibodies. In certain embodiments, neutralizing monoclonal antibodiesare chimeric monoclonal antibodies. In certain embodiments, neutralizingmonoclonal antibodies are humanized monoclonal antibodies. In certainembodiments, neutralizing monoclonal antibodies are human monoclonalantibodies. In certain embodiments, chimeric, humanized, and/or humanmonoclonal antibodies are useful as therapeutic antibodies in humans.

In certain embodiments, neutralizing monoclonal antibodies are antibodyfragments. Exemplary antibody fragments include, but are not limited to,Fab, Fab′, F(ab′)₂, Fv, scFv, Fd, diabodies, and other antibodyfragments.

In various embodiments, a neutralizing monoclonal antibody binds tohuman ANGPTL3 with a K_(D) of less than 1 μM, less than 500 nM, lessthan 300 nM, less than 200 nM, less than 100 nM, less than 75 nM, lessthan 50 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM,or less than 2 nM. In various embodiments, a neutralizing monoclonalantibody binds to a human SP1 peptide with a K_(D) of less than 1 μM,less than 500 nM, less than 300 nM, less than 200 nM, less than 100 nM,less than 75 nM, less than 50 nM, less than 30 nM, less than 25 nM, lessthan 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than4 nM, less than 3 nM, or less than 2 nM. In various embodiments, aneutralizing monoclonal antibody binds to a peptide having an amino acidsequence of SEQ ID NO: 9 with a K_(D) of less than 1 μM, less than 500nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 75nM, less than 50 nM, less than 30 nM, less than 25 nM, less than 20 nM,less than 15 nM, less than 10 nM, less than 5 nM, less than 4 nM, lessthan 3 nM, or less than 2 nM. In various embodiments, a neutralizingmonoclonal antibody binds to a peptide having an amino acid sequence ofSEQ ID NO: 10 with a K_(D) of less than 1 μM, less than 500 nM, lessthan 300 nM, less than 200 nM, less than 100 nM, less than 75 nM, lessthan 50 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM,or less than 2 nM.

In various embodiments, a neutralizing monoclonal antibody againstANGPTL3 binds to mouse ANGPTL3 (SEQ ID NO: 1) with at least 10-foldgreater affinity, at least 15-fold greater affinity, at least 20-foldgreater affinity, at least 25-fold greater affinity, at least 30-foldgreater affinity, at least 40-fold greater affinity, at least 50-foldgreater affinity, at least 100-fold greater affinity, or at least200-fold greater affinity than it binds to mouse ANGPTL4 (SEQ ID NO:107). In various embodiments, a neutralizing monoclonal antibody againstANGPTL3 binds to human ANGPTL3 (SEQ ID NO: 3) with at least 10-foldgreater affinity, at least 15-fold greater affinity, at least 20-foldgreater affinity, at least 25-fold greater affinity, at least 30-foldgreater affinity, at least 40-fold greater affinity, at least 50-foldgreater affinity, at least 100-fold greater affinity, or at least200-fold greater affinity than it binds to human ANGPTL4 (SEQ ID NO:108). In various embodiments, a neutralizing monoclonal antibody againstANGPTL3 binds to a peptide having the amino acid sequence of the mouseANGPTL3 SP1 region (SEQ ID NO: 9) with at least 10-fold greateraffinity, at least 15-fold greater affinity, at least 20-fold greateraffinity, at least 25-fold greater affinity, at least 30-fold greateraffinity, at least 40-fold greater affinity, at least 50-fold greateraffinity, at least 100-fold greater affinity, or at least 200-foldgreater affinity than it binds to a peptide having the amino acidsequence of the mouse ANGPTL4 SP1 region (SEQ ID NO: 109). In variousembodiments, a neutralizing monoclonal antibody against ANGPTL3 binds toa peptide having the amino acid sequence of the human ANGPTL3 SP1 region(SEQ ID NO: 10) with at least 10-fold greater affinity, at least 15-foldgreater affinity, at least 20-fold greater affinity, at least 25-foldgreater affinity, at least 30-fold greater affinity, at least 40-foldgreater affinity, at least 50-fold greater affinity, at least 100-foldgreater affinity, or at least 200-fold greater affinity than it binds toa peptide having the amino acid sequence of the human ANGPTL4 SP1 region(SEQ ID NO: 110).

Exemplary neutralizing monoclonal antibodies, designated 4.7.1, 4.8.3,4.9.1, 1.315.1, 5.35, and 5.50 are provided. Antibodies 4.7.1, 4.8.3,4.9.1, 5.35, and 5.50 bind to an epitope within residues 32 to 57 ofmouse ANGPTL3 or human ANGPTL3 (SEQ ID NOs: 9 and 10). In certainembodiments, antibodies 4.7.1 and 4.9.1 bind to an epitope within theSP1 region of mouse ANGPTL3 or human ANGPTL3 that includes amino acids33 to 37 and 40 of SEQ ID NO: 2 or SEQ ID NO: 3 (amino acids 2 to 6 and9 of SEQ ID NO: 9 or SEQ ID NO: 10). In certain embodiments, antibodies5.35 and 5.50 bind to an epitope within the SP1 region of mouse ANGPTL3or human ANGPTL3 that includes amino acids 34 to 37 and 40 to 42 of SEQID NO: 2 or SEQ ID NO: 3 (amino acids 3 to 6 and 9 to 11 of SEQ ID NO: 9or SEQ ID NO: 10). In certain embodiments, antibody 4.8.3 binds to anepitope within the SP1 region of mouse ANGPTL3 or human ANGPTL3 thatincludes amino acids 34 to 37, 40, and 42 of SEQ ID NO: 2 or SEQ ID NO:3 (amino acids 3 to 6, 9, and 11 of SEQ ID NO: 9 or SEQ ID NO: 10).Antibody 1.315.1 binds to an epitope within residues 42 to 116 of mouseANGPTL3 (SEQ ID NO: 61).

Antibodies 4.7.1, 4.8.3, 4.9.1, 1.315.1, 5.35, and 5.50 neutralize atleast one ANGPTL3 activity. Thus, antibodies that bind an epitope boundby at least one of antibodies 4.7.1, 4.8.3, 4.9.1, 1.315.1, 5.35, and5.50 (e.g., in either human or mouse ANGPTL3) would be expected to alsopossess neutralizing activity. Certain neutralizing monoclonalantibodies against ANGPTL3 bind to one or more peptides chosen from SEQID NOs: 9, 10, 12, 13, and 61. Certain neutralizing monoclonalantibodies against ANGPTL3 bind to one or more peptides chosen from SEQID NOs: 9 and 10. In certain embodiments, a neutralizing monoclonalantibody against ANGPTL3 binds to a peptide having the sequence of SEQID NO: 61. In certain embodiments, a neutralizing monoclonal antibodyagainst ANGPTL3 binds to a peptide having the sequence of SEQ ID NO: 12and a peptide having the sequence of SEQ ID NO: 13.

In certain embodiments, neutralizing monoclonal antibodies are providedthat bind to the same epitope to which monoclonal antibody 4.7.1 binds.In certain embodiments, neutralizing monoclonal antibodies are providedthat bind to the same epitope to which monoclonal antibody 4.8.3 binds.In certain embodiments, neutralizing monoclonal antibodies are providedthat bind to the same epitope to which monoclonal antibody 4.9.1 binds.In certain embodiments, neutralizing monoclonal antibodies are providedthat bind to the same epitope to which monoclonal antibody 5.35 binds.In certain embodiments, neutralizing monoclonal antibodies are providedthat bind to the same epitope to which monoclonal antibody 5.50 binds.In certain embodiments, neutralizing monoclonal antibodies are providedthat bind to the same epitope to which monoclonal antibody 1.315.1binds.

Certain neutralizing antibodies comprise a heavy chain comprising anamino acid sequence as set forth in SEQ ID NO: 20. Certain neutralizingantibodies comprise a heavy chain comprising an amino acid sequence asset forth in SEQ ID NO: 22. Certain neutralizing antibodies comprise aheavy chain comprising an amino acid sequence as set forth in SEQ ID NO:24. Certain neutralizing antibodies comprise a light chain comprising anamino acid sequence as set forth in SEQ ID NO: 28. Certain neutralizingantibodies comprise a light chain comprising an amino acid sequence asset forth in SEQ ID NO: 30. Certain neutralizing antibodies comprise alight chain comprising an amino acid sequence as set forth in SEQ ID NO:32.

Certain neutralizing antibodies comprise a heavy chain comprising anamino acid sequence as set forth in SEQ ID NO: 20 and a light chaincomprising an amino acid sequence as set forth in SEQ ID NO: 28. Certainneutralizing antibodies comprise a heavy chain comprising an amino acidsequence as set forth in SEQ ID NO: 22 and a light chain comprising anamino acid sequence as set forth in SEQ ID NO: 30. Certain neutralizingantibodies comprise a heavy chain comprising an amino acid sequence asset forth in SEQ ID NO: 24 and a light chain comprising an amino acidsequence as set forth in SEQ ID NO: 32.

1. Chimerized and Humanized Monoclonal Antibodies

In certain embodiments, non-human antibodies are chimerized. In certainembodiments, mouse monoclonal antibodies that specifically bind humanANGPTL3 are chimerized. Certain exemplary methods for making chimericantibodies are provided, for example, in Morrison et al. (1984) Proc.Nat'l Acad. Sci. USA 81:6851-6855; Neuberger et al. (1984) Nature312:604-608; Takeda et al. (1985) Nature 314:452-454; and U.S. Pat. Nos.6,075,181 and 5,877,397.

In certain embodiments, non-human antibodies are “humanized.” In certainembodiments, mouse monoclonal antibodies that specifically bind humanANGPTL3 are humanized. In certain embodiments, mouse monoclonalantibodies raised against mouse ANGPTL3, but which specifically bind(i.e., cross react) with human ANGPTL3, are humanized. In certainembodiments, humanized antibodies retain their binding specificity andhave reduced immunogenicity (e.g., reduced human anti-mouse antibody(HAMA) response) when administered to a human. In certain embodiments,humanization is achieved by methods including, but not limited to, CDRgrafting and human engineering, as described in detail below.

In certain embodiments of humanized antibodies, one or morecomplementarity determining regions (CDRs) from the light and heavychain variable regions of an antibody with the desired bindingspecificity (the “donor” antibody) are grafted onto human frameworkregions (FRs) in an “acceptor” antibody. Exemplary CDR grafting isdescribed, e.g., in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761,5,585,089, and 5,530,101; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86:10029-10033. In certain embodiments, one or more CDRs from the lightand heavy chain variable regions are grafted onto consensus human FRs inan acceptor antibody. To create consensus human FRs, in certainembodiments, FRs from several human heavy chain or light chain aminoacid sequences are aligned to identify a consensus amino acid sequence.

In certain embodiments, certain FR amino acids in the acceptor antibodyare replaced with FR amino acids from the donor antibody. In certainsuch embodiments, FR amino acids from the donor antibody are amino acidsthat contribute to the affinity of the donor antibody for the targetantigen. See, e.g., in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761,5,585,089, and 5,530,101; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86:10029-10033. In certain embodiments, computer programs are used formodeling donor and/or acceptor antibodies to identify residues that arelikely to be involved in binding antigen and/or to contribute to thestructure of the antigen binding site, thus assisting in the selectionof residues, such as FR residues, to be replaced in the donor antibody.

In certain embodiments, CDRs from a donor antibody are grafted onto anacceptor antibody comprising a human constant region. In certain suchembodiments, FRs are also grafted onto the acceptor. In certainembodiments, CDRs from a donor antibody are derived from a single chainFv antibody. In certain embodiments, FRs from a donor antibody arederived from a single chain Fv antibody. In certain embodiments, graftedCDRs in a humanized antibody are further modified (e.g., by amino acidsubstitutions, deletions, or insertions) to increase the affinity of thehumanized antibody for the target antigen. In certain embodiments,grafted FRs in a humanized antibody are further modified (e.g., by aminoacid substitutions, deletions, or insertions) to increase the affinityof the humanized antibody for the target antigen.

In certain embodiments, non-human antibodies may be humanized using a“human engineering” method. See, e.g., U.S. Pat. Nos. 5,766,886 and5,869,619. In certain embodiments of human engineering, information onthe structure of antibody variable domains (e.g., information obtainedfrom crystal structures and/or molecular modeling) is used to assess thelikelihood that a given amino acid residue in a variable region is (a)involved in antigen binding, (b) exposed on the antibody surface (i.e.,accessible to solvent), or (c) buried within the antibody variableregion (i.e., involved in maintaining the structure of the variableregion). Furthermore, in certain embodiments, human variable regionconsensus sequences are generated to identify residues that areconserved among human variable regions. In certain embodiments, thatinformation provides guidance as to whether an amino acid residue in thevariable region of a non-human antibody should be substituted.

Certain neutralizing antibodies comprise a heavy chain comprising CDR1,CDR2, and CDR3 of 4.7.1. Certain neutralizing antibodies comprise aheavy chain comprising CDR1, CDR2, and CDR3 of 4.8.3. Certainneutralizing antibodies comprise a heavy chain comprising CDR1, CDR2,and CDR3 of 4.9.1. Certain neutralizing antibodies comprise a heavychain comprising CDR1, CDR2, and CDR3 of 5.35. Certain neutralizingantibodies comprise a heavy chain comprising CDR1, CDR2, and CDR3 of5.50. Certain neutralizing antibodies comprise a heavy chain comprisingCDR1, CDR2, and CDR3 of 1.315.1. Certain neutralizing antibodiescomprise a heavy chain comprising at least one CDR of 4.7.1. Certainneutralizing antibodies comprise a heavy chain comprising at least oneCDR of 4.8.3. Certain neutralizing antibodies comprise a heavy chaincomprising at least one CDR of 4.9.1. Certain neutralizing antibodiescomprise a heavy chain comprising at least one CDR of 5.35. Certainneutralizing antibodies comprise a heavy chain comprising at least oneCDR of 5.50. Certain neutralizing antibodies comprise a heavy chaincomprising at least one CDR of 1.315.1. Certain neutralizing antibodiescomprise a heavy chain comprising at least two CDRs of 4.7.1. Certainneutralizing antibodies comprise a heavy chain comprising at least twoCDRs of 4.8.3. Certain neutralizing antibodies comprise a heavy chaincomprising at least two CDRs of 4.9.1. Certain neutralizing antibodiescomprise a heavy chain comprising at least two CDRs of 5.35. Certainneutralizing antibodies comprise a heavy chain comprising at least twoCDRs of 5.50. Certain neutralizing antibodies comprise a heavy chaincomprising at least two CDRs of 1.315.1.

Certain neutralizing antibodies comprise a light chain comprising CDR1,CDR2, and CDR3 of 4.7.1. Certain neutralizing antibodies comprise alight chain comprising CDR1, CDR2, and CDR3 of 4.8.3. Certainneutralizing antibodies comprise a light chain comprising CDR1, CDR2,and CDR3 of 4.9.1. Certain neutralizing antibodies comprise a lightchain comprising CDR1, CDR2, and CDR3 of 5.35. Certain neutralizingantibodies comprise a light chain comprising CDR1, CDR2, and CDR3 of5.50. Certain neutralizing antibodies comprise a light chain comprisingCDR1, CDR2, and CDR3 of 1.315.1. Certain neutralizing antibodiescomprise a light chain comprising at least one CDR of 4.7.1. Certainneutralizing antibodies comprise a light chain comprising at least oneCDR of 4.8.3. Certain neutralizing antibodies comprise a light chaincomprising at least one CDR of 4.9.1. Certain neutralizing antibodiescomprise a light chain comprising at least one CDR of 5.35. Certainneutralizing antibodies comprise a light chain comprising at least oneCDR of 5.50. Certain neutralizing antibodies comprise a light chaincomprising at least one CDR of 1.315.1. Certain neutralizing antibodiescomprise a light chain comprising at least two CDRs of 4.7.1. Certainneutralizing antibodies comprise a light chain comprising at least twoCDRs of 4.8.3. Certain neutralizing antibodies comprise a light chaincomprising at least two CDRs of 4.9.1. Certain neutralizing antibodiescomprise a light chain comprising at least two CDRs of 5.35. Certainneutralizing antibodies comprise a light chain comprising at least twoCDRs of 5.50. Certain neutralizing antibodies comprise a light chaincomprising at least two CDRs of 1.315.1.

2. Antibody Isotypes

In certain embodiments, an antibody against ANGPTL3 is of any isotypeselected from IgM, IgD, IgG, IgA, and IgE. In certain embodiments, anantibody against ANGPTL3 is of the IgG isotype. In certain suchembodiments, an antibody is of the subclass IgG1, IgG2, IgG3, or IgG4.In certain embodiments, an antibody against ANGPTL3 is of the IgMisotype. In certain such embodiments, an antibody is of the subclassIgM1 or IgM2. In certain embodiments, an antibody against ANGPTL3 is ofthe IgA isotype. In certain such embodiments, an antibody is of thesubclass IgA1 or IgA2. In certain embodiments, an antibody againstANGPTL3 comprises a human kappa light chain and a human IgG1 or IgG2heavy chain. In certain embodiments, an antibody against ANGPTL3comprises a mouse kappa light chain and a mouse IgG1 or IgG2 heavychain.

3. Modified Antibodies

In various embodiments, an antibody is modified to alter one or more ofits properties. In certain embodiments, a modified antibody may possessadvantages over an unmodified antibody, such as increased stability,increased time in circulation, or decreased immunogenicity (see, e.g.,U.S. Pat. No. 4,179,337). In certain embodiments, an antibody ismodified by linking it to a nonproteinaceous moiety. In certainembodiments, an antibody is modified by altering the glycosylation stateof the antibody, e.g., by altering the number, type, linkage, and/orposition of carbohydrate chains on the antibody. In certain embodiments,an antibody is altered so that it is not glycosylated.

In certain embodiments, one or more chemical moieties are linked to theamino acid backbone and/or carbohydrate residues of the antibody.Certain exemplary methods for linking a chemical moiety to an antibodyare known to those skilled in the art. Such methods include, but are notlimited to, acylation reactions or alkylation reactions. See, e.g, EP 0401 384; Malik et al. (1992), Exp. Hematol., 20:1028-1035; Francis(1992) Focus on Growth Factors 3(2):4-10, published by Mediscript,Mountain Court, Friern Barnet Lane, London N20 OLD, UK; EP 0 154 316; EP0 401 384; WO 92/16221; WO 95/34326; WO 95/13312; WO 96/11953; WO96/19459 and WO 96/19459. In certain embodiments, any of these reactionsare used to generate an antibody that is chemically modified at itsamino-terminus.

In certain embodiments, an antibody is linked to a detectable label,such as an enzymatic, fluorescent, isotopic or affinity label. Incertain such embodiments, a detectable label allows for the detection orisolation of the antibody. In certain embodiments, a detectable labelallows for the detection of an antigen bound by the antibody.

In certain embodiments, an antibody is modified by linking it to one ormore polymers. In certain embodiments, an antibody is linked to one ormore water-soluble polymers. In certain such embodiments, linkage to awater-soluble polymer reduces the likelihood that the antibody willprecipitate in an aqueous environment, such as a physiologicalenvironment. In certain embodiments, a therapeutic antibody is linked toa water-soluble polymer. In certain embodiments, one skilled in the artcan select a suitable water-soluble polymer based on considerationsincluding, but not limited to, whether the polymer/antibody conjugatewill be used in the treatment of a patient and, if so, thepharmacological profile of the antibody (e.g., half-life, dosage,activity, antigenicity, and/or other factors).

Certain exemplary clinically acceptable, water-soluble polymers include,but are not limited to, polyethylene glycol (PEG); polyethylene glycolpropionaldehyde; copolymers of ethylene glycol/propylene glycol;monomethoxy-polyethylene glycol; carboxymethylcellulose; dextran;polyvinyl alcohol (PVA); polyvinyl pyrrolidone, poly-1,3-dioxolane;poly-1,3,6-trioxane; ethylene/maleic anhydride copolymer; poly-β-aminoacids (either homopolymers or random copolymers); poly(n-vinylpyrrolidone)polyethylene glycol; polypropylene glycol homopolymers (PPG)and other polyalkylene oxides; polypropylene oxide/ethylene oxidecopolymers; polyoxyethylated polyols (POG) (e.g., glycerol) and otherpolyoxyethylated polyols; polyoxyethylated sorbitol, polyoxyethylatedglucose, colonic acids or other carbohydrate polymers; and Ficoll,dextran, or mixtures thereof. Certain exemplary PEGs include, but arenot limited to, certain forms known in the art to be useful in antibodymodification, such as mono-(C₁-C₁₀) alkoxy- or aryloxy-PEG. In certainembodiments, PEG propionaldehyde may have advantages in manufacturingdue to its stability in water.

In certain embodiments, a water-soluble polymer is of any molecularweight. In certain embodiments, a water-soluble polymer is branched orunbranched. In certain embodiments, a water-soluble polymer has anaverage molecular weight of about 2 kDa to about 100 kDa, including allpoints between the end points of the range. In certain embodiments, awater-soluble polymer has an average molecular weight of about 5 kDa toabout 40 kDa. In certain embodiments, a water-soluble polymer has anaverage molecular weight of about 10 kDa to about 35 kDa. In certainembodiments, a water-soluble polymer has an average molecular weight ofabout 15 kDa to about 30 kDa.

In certain embodiments, an antibody is linked to PEG (i.e., an antibodyis “pegylated”). In various embodiments, PEG has low toxicity inmammals. See Carpenter et al. (1971) Toxicol. Appl. Pharmacol.,18:35-40. Notably, a PEG adduct of adenosine deaminase was approved inthe United States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. In various embodiments, PEG may reduce theimmunogenicity of antibodies. For example, in certain embodiments,linkage of PEG to an antibody having non-human sequences may reduce theantigenicity of that antibody when administered to a human.

In certain embodiments, a polymer is linked to one or more reactiveamino acid residues in an antibody. Certain exemplary reactive aminoacid residues include, but are not limited to, the alpha-amino group ofthe amino-terminal amino acid, the epsilon amino groups of lysine sidechains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, and activatedglycosyl chains linked to certain asparagine, serine or threonineresidues. Certain exemplary activated forms of PEG (“PEG reagents”)suitable for direct reaction with proteins are known to those skilled inthe art. For example, in certain embodiments, PEG reagents suitable forlinkage to amino groups include, but are not limited to, active estersof carboxylic acid or carbonate derivatives of PEG, for example, thosein which the leaving groups are N-hydroxysuccinimide, p-nitrophenol,imidazole or 1-hydroxy-2-nitrobenzene-4-sulfonate. In certainembodiments, PEG reagents containing maleimido or haloacetyl groups areused to modify sulfhydryl groups. In certain embodiments, PEG reagentscontaining amino, hydrazine and/or hydrazide groups may be used inreactions with aldehydes generated by periodate oxidation ofcarbohydrate groups in proteins.

In certain embodiments, a water-soluble polymer has at least onereactive group. In certain embodiments, an activated derivative of awater-soluble polymer, such as PEG, is created by reacting thewater-soluble polymer with an activating group. In certain embodiments,an activating group may be monofunctional, bifunctional, ormultifunctional. Certain exemplary activating groups that can be used tolink a water-soluble polymer to two or more antibodies include, but arenot limited to, the following groups: sulfone (e.g., chlorosulfone,vinylsulfone and divinylsulfone), maleimide, sulfhydryl, thiol,triflate, tresylate, azidirine, oxirane and 5-pyridyl. In certainembodiments, a PEG derivative is typically stable against hydrolysis forextended periods in aqueous environments at pHs of about 11 or less. Incertain embodiments, a PEG derivative linked to another molecule, suchas an antibody, confers stability from hydrolysis on that molecule.Certain exemplary homobifunctional PEG derivatives include, but are notlimited to, PEG-bis-chlorosulfone and PEG-bis-vinylsulfone (see WO95/13312).

D. Certain Methods of Making Monoclonal Antibodies

1. Certain Hybridoma Methods

In certain embodiments, monoclonal antibodies are produced by standardtechniques. In certain embodiments, monoclonal antibodies are producedby hybridoma-based methods. Certain such methods are known to thoseskilled in the art. See, e.g., Kohler et al. (1975) Nature 256:495-497;Harlow and Lane (1988) Antibodies: A Laboratory Manual Ch. 6 (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.). In certain suchembodiments, a suitable animal, such as a mouse, rat, hamster, monkey,or other mammal, is immunized with an immunogen to produceantibody-secreting cells. In certain embodiments, the antibody-secretingcells are B-cells, such as lymphocytes or splenocytes. In certainembodiments, lymphocytes (e.g., human lymphocytes) are immunized invitro to generate antibody-secreting cells. See, e.g., Borreback et al.(1988) Proc. Nat'l Acad. Sci. USA 85:3995-3999.

In certain embodiments, antibody secreting cells are fused with an“immortalized” cell line, such as a myeloid-type cell line, to producehybridoma cells. In certain embodiments, hybridoma cells that producethe desired antibodies are identified, for example, by ELISA. In certainembodiments, such cells can then be subcloned and cultured usingstandard methods. In certain embodiments, such cells can also be grownin vivo as ascites tumors in a suitable animal host. In certainembodiments, monoclonal antibodies are isolated from hybridoma culturemedium, serum, or ascites fluid using standard separation procedures,such as affinity chromatography. Guidance for the production ofhybridomas and the purification of monoclonal antibodies according tocertain embodiments is provided, for example, in Harlow and Lane (1988)Antibodies: A Laboratory Manual Ch. 8 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

In certain embodiments, mouse monoclonal antibodies are produced byimmunizing genetically altered mice with an immunogen. In certain suchembodiments, the mice are ANGPTL3-deficient mice, which partially orcompletely lack ANGPTL3 function. In certain such embodiments, the miceare “knockout” mice that lack all or part of a gene encoding ANGPTL3. Incertain embodiments, such knockout mice are immunized with mouseANGPTL3. In certain embodiments, such knockout mice are immunized withhuman ANGPTL3.

In certain embodiments, human monoclonal antibodies are raised intransgenic animals (e.g., mice) that are capable of producing humanantibodies. See, e.g., U.S. Pat. Nos. 6,075,181 A and 6,114,598 A; andWO 98/24893 A2. For example, in certain embodiments, humanimmunoglobulin genes are introduced (e.g., using yeast artificialchromosomes, human chromosome fragments, or germline integration) intomice in which the endogenous Ig genes have been inactivated. See, e.g.,Jakobovits et al. (1993) Nature 362:255-258; Tomizuka et al. (2000)Proc. Nat'l Acad. Sci. USA 97:722-727; and Mendez et al. (1997) Nat.Genet. 15:146-156 (describing the XenoMouse II® line of transgenicmice).

In certain embodiments, such transgenic mice are immunized with animmunogen. In certain such embodiments, lymphatic cells (such asB-cells) from mice that express antibodies are obtained. In certain suchembodiments, such recovered cells are fused with an “immortalized” cellline, such as a myeloid-type cell line, to produce hybridoma cells. Incertain such embodiments, hybridoma cells are screened and selected toidentify those that produce antibodies specific to the antigen ofinterest. Certain exemplary methods and transgenic mice suitable for theproduction of human monoclonal antibodies are described, e.g., inJakobovits et al. (1993) Nature 362:255-258; Jakobovits (1995) Curr.Opin. Biotechnol. 6:561-566; Lonberg et al. (1995) Int'l Rev. Immunol.13:65-93; Fishwild et al. (1996) Nat. Biotechnol. 14:845-851; Mendez etal. (1997) Nat. Genet. 15:146-156; Green (1999) J. Immunol. Methods231:11-23; Tomizuka et al. (2000) Proc. Nat'l Acad. Sci. USA 97:722-727;and reviewed in Little et al. (2000) Immunol. Today 21:364-370; and WO98/24893. In certain embodiments, human monoclonal antibodies againstANGPTL3 are suitable for use as therapeutic antibodies. See Part V.G.,below.

2. Certain Display-Based Methods

In certain embodiments, human monoclonal antibodies are produced using adisplay-based method, such as, for example, any of those describedbelow.

In certain embodiments, a monoclonal antibody is produced using phagedisplay techniques. Certain exemplary antibody phage display methods areknown to those skilled in the art and are described, for example, inHoogenboom, Overview of Antibody Phage-Display Technology and ItsApplications, from Methods in Molecular Biology: Antibody Phage Display:Methods and Protocols (2002) 178:1-37 (O'Brien and Aitken, eds., HumanPress, Totowa, N.J.). For example, in certain embodiments, a library ofantibodies are displayed on the surface of a filamentous phage, such asthe nonlytic filamentous phage fd or M13. In certain embodiments, theantibodies are antibody fragments, such as scFvs, Fabs, Fvs with anengineered intermolecular disulfide bond to stabilize the V_(H)-V_(L)pair, and diabodies. In certain embodiments, antibodies with the desiredbinding specificity can then be selected. Certain exemplary embodimentsof antibody phage display methods are described in further detail below.

In certain embodiments, an antibody phage-display library can beprepared using certain methods known to those skilled in the art. See,e.g., Hoogenboom, Overview of Antibody Phage-Display Technology and ItsApplications, from Methods in Molecular Biology: Antibody Phage Display:Methods and Protocols (2002) 178:1-37 (O'Brien and Aitken, eds., HumanPress, Totowa, N.J.). In certain embodiments, variable gene repertoiresare prepared by PCR amplification of genomic DNA or cDNA derived fromthe mRNA of antibody-secreting cells. For example, in certainembodiments, cDNA is prepared from mRNA of B-cells. In certainembodiments, cDNA encoding the variable regions of heavy and lightchains is amplified, for example, by PCR.

In certain embodiments, heavy chain cDNA and light chain cDNA are clonedinto a suitable vector. In certain embodiments, heavy chain cDNA andlight chain cDNA are randomly combined during the cloning process,thereby resulting in the assembly of a cDNA library encoding diversescFvs or Fabs. In certain embodiments, heavy chain cDNA and light chaincDNA are ligated before being cloned into a suitable vector. In certainembodiments, heavy chain cDNA and light chain cDNA are ligated bystepwise cloning into a suitable vector.

In certain embodiments, cDNA is cloned into a phage display vector, suchas a phagemid vector. Certain exemplary phagemid vectors, such as pCES1,are known to those skilled in the art. In certain embodiments, cDNAencoding both heavy and light chains is present on the same vector. Forexample, in certain embodiments, cDNA encoding scFvs are cloned in framewith all or a portion of gene III, which encodes the minor phage coatprotein pIII. In certain such embodiments, the phagemid directs theexpression of the scFv-pIII fusion on the phage surface. Alternatively,in certain embodiments, cDNA encoding heavy chain (or light chain) iscloned in frame with all or a portion of gene III, and cDNA encodinglight chain (or heavy chain) is cloned downstream of a signal sequencein the same vector. The signal sequence directs expression of the lightchain (or heavy chain) into the periplasm of the host cell, where theheavy and light chains assemble into Fab fragments. Alternatively, incertain embodiments, cDNA encoding heavy chain and cDNA encoding lightchain are present on separate vectors. In certain such embodiments,heavy chain and light chain cDNA is cloned separately, one into aphagemid and the other into a phage vector, which both contain signalsfor in vivo recombination in the host cell.

In certain embodiments, recombinant phagemid or phage vectors areintroduced into a suitable bacterial host, such as E. coli. In certainembodiments using phagemid, the host is infected with helper phage tosupply phage structural proteins, thereby allowing expression of phageparticles carrying the antibody-pIII fusion protein on the phagesurface.

In certain embodiments, “synthetic” antibody libraries are constructedusing repertoires of variable genes that are rearranged in vitro. Forexample, in certain embodiments, individual gene segments encoding heavyor light chains (V-D-J or V-J, respectively) are randomly combined usingPCR. In certain such embodiments, additional sequence diversity can beintroduced into the CDRs, and possibly FRs, e.g., by error prone PCR. Incertain such embodiments, additional sequence diversity is introducedinto CDR3, e.g., H3 of the heavy chain.

In certain embodiments, “naïve” or “universal” phage display librariesare constructed as described above using nucleic acid from anunimmunized animal. In certain embodiments, the unimmunized animal is ahuman. In certain embodiments, “immunized” phage display libraries areconstructed as described above using nucleic acid from an immunizedanimal. In certain embodiments, the immunized animal is a human, rat,mouse, hamster, or monkey. In certain such embodiments, the animals areimmunized with any of the immunogens described below.

Certain exemplary universal human antibody phage display libraries areavailable from commercial sources. Certain exemplary libraries include,but are not limited to, the HuCAL® series of libraries from MorphoSys AG(Martinstreid/Munich, Germany); libraries from Crucell (Leiden, theNetherlands) using MAbstract® technology; the n-CoDeR™ Fab library fromBioInvent (Lund, Sweden); and libraries available from CambridgeAntibody Technology (Cambridge, UK).

In certain embodiments, the selection of antibodies having the desiredbinding specificity from a phage display library is achieved bysuccessive panning steps. In certain embodiments of panning, libraryphage preparations are exposed to antigen. In certain such embodiments,the phage-antigen complexes are washed, and unbound phage are discarded.In certain such embodiments, bound phage are recovered and subsequentlyamplified by infecting E. coli. In certain such embodiments, monoclonalantibody-producing phage may be cloned by picking single plaques. Incertain embodiments, the above process is repeated.

In certain embodiments, the antigen used in panning is any of theimmunogens described below. In certain embodiments, the antigen isimmobilized on a solid support to allow purification of antigen-bindingphage by affinity chromatography. In certain embodiments, the antigen isbiotinylated, thereby allowing the separation of bound phage fromunbound phage using streptavidin-coated magnetic beads. In certainembodiments, the antigen may be immobilized on cells (for directpanning), in tissue cryosections, or on membranes (e.g., nylon ornitrocellulose membranes). Other variations of certain panningprocedures may be routinely determined by one skilled in the art.

In certain embodiments, a yeast display system is used to producemonoclonal antibodies. In certain such systems, an antibody is expressedas a fusion protein with all or a portion of the yeast AGA2 protein,which becomes displayed on the surface of the yeast cell wall. Incertain such embodiments, yeast cells expressing antibodies with thedesired binding specificity can then be identified by exposing the cellsto fluorescently labeled antigen. In certain such embodiments, yeastcells that bind the antigen can then be isolated by flow cytometry. See,e.g., Boder et al. (1997) Nat. Biotechnol. 15:553-557.

3. Certain Affinity Maturation Methods

In certain embodiments, the affinity of an antibody for a particularantigen is increased by subjecting the antibody to affinity maturation(or “directed evolution”) in vitro. In vivo, native antibodies undergoaffinity maturation through somatic hypermutation followed by selection.Certain in vitro methods mimic that in vivo process, thereby allowingthe production of antibodies having affinities that equal or surpassthat of native antibodies.

In certain embodiments of affinity maturation, mutations are introducedinto a nucleic acid sequence encoding the variable region of an antibodyhaving the desired binding specificity. See, e.g., Hudson et al. (2003)Nat. Med. 9:129-134; Brekke et al. (2002) Nat. Reviews 2:52-62. Incertain embodiments, mutations are introduced into the variable regionof the heavy chain, light chain, or both. In certain embodiments,mutations are introduced into one or more CDRs. In certain suchembodiments, mutations are introduced into H3, L3, or both. In certainembodiments, mutations are introduced into one or more FRs. In certainembodiments, a library of mutations is created, for example, in a phage,ribosome, or yeast display library, so that antibodies with increasedaffinity may be identified by standard screening methods. See, e.g.,Boder et al. (2000) Proc. Nat'l Acad. Sci. USA 97:10701-10705; Foote etal. (2000) Proc. Nat'l Acad. Sci. USA 97:10679-10681; Hoogenboom,Overview of Antibody Phage-Display Technology and Its Applications, fromMethods in Molecular Biology: Antibody Phage Display: Methods andProtocols (2002) 178:1-37 (O'Brien and Aitken, eds., Human Press,Totowa, N.J.); and Hanes et al. (1998) Proc. Nat'l Acad. Sci. USA95:14130-14135.

In certain embodiments, mutations are introduced by site-specificmutagenesis based on information on the antibody's structure, e.g., theantigen binding site. In certain embodiments, mutations are introducedusing combinatorial mutagenesis of CDRs. In certain embodiments, all ora portion of the variable region coding sequence is randomlymutagenized, e.g., using E. coli mutator cells, homologous generearrangement, or error prone PCR. In certain embodiments, mutations areintroduced using “DNA shuffling.” See, e.g., Crameri et al. (1996) Nat.Med. 2:100-102; Fermer et al. (2004) Tumor Biol. 25:7-13.

In certain embodiments, “chain shuffling” is used to generate antibodieswith increased affinity. In certain embodiments of chain shuffling, oneof the chains, e.g., the light chain, is replaced with a repertoire oflight chains, while the other chain, e.g., the heavy chain, isunchanged, thus providing specificity. In certain such embodiments, alibrary of chain shuffled antibodies is created, wherein the unchangedheavy chain is expressed in combination with each light chain from therepertoire of light chains. In certain embodiments, such libraries maythen be screened for antibodies with increased affinity. In certainembodiments, both the heavy and light chains are sequentially replaced.In certain embodiments, only the variable regions of the heavy and/orlight chains are replaced. In certain embodiments, only a portion of thevariable regions, e.g., CDRs, of the heavy and/or light chains arereplaced. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; Brekke etal. (2002) Nat. Reviews 2:52-62; Kang et al. (1991) Proc. Nat'l Acad.Sci. USA 88:11120-11123; Marks et al. (1992) Biotechnol. 10:779-83.

In certain embodiments, mouse monoclonal antibodies that specificallybind human ANGPTL3 (including, but not limited to, mouse monoclonalantibodies raised against mouse ANGPTL3 but which specifically bind(i.e., cross react) with human ANGPTL3) are subject to sequential chainshuffling. In certain embodiments, for example, the heavy chain of agiven mouse monoclonal antibody is combined with a new repertoire ofhuman light chains, and antibodies with the desired affinity areselected. In certain such embodiments, the light chains of the selectedantibodies are then combined with a new repertoire of human heavychains, and antibodies with the desired affinity are selected. Thus, incertain embodiments, human antibodies having the desired antigen bindingspecificity and affinity are selected.

Alternatively, in certain embodiments, the heavy chain of a given mousemonoclonal antibody is combined with a new repertoire of human lightchains, and antibodies with the desired affinity are selected from thisfirst round of shuffling. In certain embodiments, the light chain of theoriginal mouse monoclonal antibody is combined with a new repertoire ofhuman heavy chains, and antibodies with the desired affinity areselected from this second round of shuffling. In certain embodiments,human light chains from the antibodies selected in the first round ofshuffling are then combined with human heavy chains from the antibodiesselected in the second round of shuffling. Thus, in certain embodiments,human antibodies having the desired antigen binding specificity andaffinity are selected.

In certain embodiments, a “ribosome display” method is used thatalternates antibody selection with affinity maturation. In certainembodiments of a ribosome display method, antibody-encoding nucleic acidis amplified by RT-PCR between the selection steps. Thus, in certainembodiments, error prone polymerases may be used to introduce mutationsinto the nucleic acid. A nonlimiting example of such a method isdescribed in detail in Hanes et al. (1998) Proc. Nat'l Acad. Sci. USA95:14130-14135.

4. Certain Recombinant Methods

In certain embodiments, a monoclonal antibody is produced by recombinanttechniques. See, e.g., U.S. Pat. No. 4,816,567. In certain suchembodiments, nucleic acid encoding monoclonal antibody chains are clonedand expressed in a suitable host cell. For example, in certainembodiments, RNA can be prepared from cells expressing the desiredantibody, such as mature B-cells or hybridoma cells, using standardmethods. In certain embodiments, the RNA can then be used to make cDNAusing standard methods. In certain embodiments, cDNA encoding a heavy orlight chain polypeptide is amplified, for example, by PCR, usingspecific oligonucleotide primers. In certain embodiments, the cDNA iscloned into a suitable expression vector. In certain embodiments, theexpression vector is then transformed or transfected into a suitablehost cell, such as a host cell that does not endogenously produceantibody. Certain exemplary host cells include, but are not limited to,E. coli, COS cells, Chinese hamster ovary (CHO) cells, and myelomacells. In certain embodiments, wherein heavy and light chains arecoexpressed in the same host, reconstituted antibody may be isolated.

In certain embodiments, cDNA encoding a heavy or light chain can bemodified. For example, in certain embodiments, the constant region of amouse heavy or light chain can be replaced with the constant region of ahuman heavy or light chain. In this manner, in certain embodiments, achimeric antibody can be produced which possesses human antibodyconstant regions but retains the binding specificity of a mouseantibody.

In certain embodiments, recombinant antibodies can be expressed incertain cell lines. In certain embodiments, sequences encodingparticular antibodies can be used for transformation of a suitablemammalian host cell. According to certain embodiments, transformationcan be by any known method for introducing polynucleotides into a hostcell. Certain exemplary methods include, but are not limited to,packaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) and using certaintransfection procedures known in the art, as exemplified by U.S. Pat.Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. In certainembodiments, the transformation procedure used may depend upon the hostto be transformed. Certain exemplary methods for introduction ofheterologous polynucleotides into mammalian cells are known in the artand include, but are not limited to, dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Certain exemplary mammalian cell lines available as hosts for expressionare known in the art and include, but are not limited to, manyimmortalized cell lines available from the American Type CultureCollection (ATCC), including but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and anumber of other cell lines. In certain embodiments, cell lines may beselected by determining which cell lines produce high levels ofantibodies that specifically bind ANGPTL3.

E. Certain Polypeptide Immunogens

In certain embodiments, to generate antibodies, an animal is immunizedwith an immunogen. In certain embodiments, an immunogen is a polypeptidecomprising ANGPTL3. In certain embodiments, an immunogen is apolypeptide comprising a fragment of ANGPTL3. In certain embodiments, animmunogen is a polypeptide comprising the N-terminal coiled-coil domainof ANGPTL3. In certain embodiments, an immunogen is a polypeptidecomprising the SP1 region of ANGPTL3.

In certain embodiments, an immunogen comprises a mouse ANGPTL3. Incertain embodiments, an immunogen comprises a human ANGPTL3. In certainembodiments, an immunogen comprises a mouse ANGPTL3 comprising the aminoacid sequence of SEQ ID NO: 1. In certain embodiments, an immunogencomprises a human ANGPTL3 comprising the amino acid sequence of SEQ IDNO: 3. In certain embodiments, an immunogen comprises a fragment ofmouse ANGPTL3. In certain embodiments, an immunogen comprises a fragmentof SEQ ID NO: 1 from residue 17 to residue 240. In certain embodiments,an immunogen comprises the amino acid sequence of SEQ ID NO: 59. Incertain embodiments, an immunogen comprises a fragment of SEQ ID NO: 1from residue 32 to residue 57. In certain embodiments, an immunogencomprises the amino acid sequence of SEQ ID NO: 9. In certainembodiments, an immunogen comprises the amino acid sequence of SEQ IDNO: 61. In certain embodiments, an immunogen comprises a fragment ofhuman ANGPTL3. In certain embodiments, an immunogen comprises a fragmentof SEQ ID NO: 3 from residue 20 to residue 243. In certain embodiments,an immunogen comprises the amino acid sequence of SEQ ID NO: 60. Incertain embodiments, an immunogen comprises a fragment of SEQ ID NO: 1from residue 32 to residue 57. In certain embodiments, an immunogencomprises the amino acid sequence of SEQ ID NO: 10.

In certain embodiments, an immunogen comprises any peptide of about10-20 contiguous amino acids from residue 17 to residue 240 of SEQ IDNO: 1. In certain embodiments, an immunogen comprises any peptide ofabout 10-20 contiguous amino acids from residue 20 to residue 243 of SEQID NO: 3. In certain embodiments, an immunogen comprises one or morepeptides selected from SEQ ID NOs: 9, 10, 12, 13, 59, 60, and 61. Incertain embodiments, an immunogen comprises a peptide selected from SEQID NOs: 9 and 10. In certain embodiments, an immunogen comprises apeptide comprising one or more amino acid sequences selected from SEQ IDNOs: 59 and 60. In certain embodiments, an immunogen comprises a peptidecomprising SEQ ID NOs: 12, 13, and 61. In certain such embodiments, apeptide is selected that is likely to be immunogenic. In certain suchembodiments, a peptide is selected that is predicted to be hydrophilicand/or likely to be exposed on the surface of native ANGPTL3 in itsfolded state. Exemplary guidance for selecting suitable immunogenicpeptides is provided, for example, in Ausubel et al. (1989) CurrentProtocols in Molecular Biology Ch. 11.14 (John Wiley & Sons, NY); andHarlow and Lane (1988) Antibodies: A Laboratory Manual Ch. 5 (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Certain exemplary algorithms are known to those skilled in the art forpredicting whether a peptide segment of a protein is hydrophilic andtherefore likely to be exposed on the surface of the protein. Certainsuch algorithms use the primary sequence information of a protein tomake such predictions. Certain such algorithms are based on the methodof, for example, Hopp and Woods (1981) Proc. Nat'l Acad. Sci. USA78:3824-3828, or Kyte and Doolittle (1982) J. Mol. Biol. 157:105-132.Certain exemplary algorithms are known to those skilled in the art forpredicting the secondary structure of a protein based on the primaryamino acid sequence of the protein. See, e.g., Corrigan et al. (1982)Comput. Programs Biomed. 3:163-168. Certain such algorithms are based onthe method of, for example, Chou and Fasman (1978) Ann. Rev. Biochem.47:25-276. In certain embodiments, peptide segments that are predictedto form β-turns, and are therefore likely to be exposed on the surfaceof a protein, may be selected as immunogens.

In certain embodiments, an animal is immunized with an immunogen and oneor more adjuvants. In certain embodiments, an adjuvant is used toincrease the immunological response, depending on the host species.Certain exemplary adjuvants include, but are not limited to, Freund'sadjuvant (complete and incomplete), mineral salts such as aluminumhydroxide or aluminum phosphate, surface active substances, chitosan,lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum. In certain embodiments, the immune responseto an immunogen, e.g., a peptide immunogen, is enhanced by coupling theimmunogen to another immunogenic molecule or “carrier protein.” Certainexemplary carrier proteins include, but are not limited to, keyholelimpet hemocyanin (KLH), tetanus toxoid, diphtheria toxoid, ovalbumin,cholera toxoid, and immunogenic fragments thereof. For exemplaryguidance in coupling peptide immunogens to carrier proteins, see, e.g.,Ausubel et al. (1989) Current Protocols in Molecular Biology Ch. 11.15(John Wiley & Sons, NY); and Harlow and Lane (1988) Antibodies: ALaboratory Manual Ch. 5 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

In certain embodiments, any of the above immunogens can be producedusing standard recombinant methods. For example, in certain embodiments,a polynucleotide encoding a mouse or human ANGPTL3 or a fragment of thatpolynucleotide may be cloned into a suitable expression vector. Incertain embodiments, the polynucleotide comprises the nucleic acidsequence of SEQ ID NO: 5 or SEQ ID NO: 6. In certain embodiments, therecombinant vector is then introduced into a suitable host cell. Incertain embodiments, the polypeptide is then isolated from the host cellby standard methods. For certain exemplary methods of recombinantprotein expression, see, e.g., Ausubel et al. (1991) Current Protocolsin Molecular Biology Ch. 16 (John Wiley & Sons, NY).

F. Certain Assays

1. Certain Binding Assays

In certain embodiments, antibodies are screened for binding to ANGPTL3using certain routine methods that detect binding of antibody toantigen. For example, in certain embodiments, the ability of amonoclonal antibody to bind ANGPTL3 is assayed by standardimmunoblotting methods, such as Western blot. See, e.g., Ausubel et al.(1992) Current Protocols in Molecular Biology Ch. 10.8 (John Wiley &Sons, NY); Harlow and Lane (1988) Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.). In certainembodiments, ANGPTL3 to be used in such assays may be isolated or may bepresent in a complex mixture of proteins and/or macromolecules.

In certain embodiments, the ability of a monoclonal antibody to bindANGPTL3 is assayed using a competitive binding assay, which evaluatesthe ability of a candidate antibody to compete with a known anti-ANGPTL3antibody for binding to ANGPTL3. In certain such embodiments, the knownanti-ANGPTL3 antibody is any of the monoclonal antibodies describedbelow in Part VI.J. In certain embodiments, a competitive binding assayis performed using ELISA. See, e.g., Harlow and Lane (1988) Antibodies:A Laboratory Manual Ch. 14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

In certain embodiments, a binding assay is used to quantify the bindingkinetics (e.g., rate constant) or the binding affinity (e.g.,association or dissociation constant) of an antibody against ANGPTL3. Incertain embodiments, the kinetics or affinity of binding is determinedin the “solid-phase” by immobilizing antigen (e.g., ANGPTL3) on a solidsupport. The immobilized antigen “captures” antibody from solution. Incertain embodiments, the kinetics or affinity of binding is determinedin the “solid-phase”by immobilizing antibody (e.g., antibody againstANGPTL3) on a solid support. The immobilized antibody “captures” antigenfrom solution.

In certain embodiments, binding kinetics or binding affinity isdetermined using ELISA-based methods. In certain embodiments, bindingkinetics or binding affinity is determined using biosensor-basedtechnology, such as Biacore surface plasmon resonance technology(Biacore, Piscataway, N.J.). Certain such methods are known to thoseskilled in the art. See, e.g., McCafferty et al. (eds.) (1996) AntibodyEngineering: A Practical Approach (IRL, Oxford, UK); Goldberg et al.(1993) Curr. Opin. Immunol. 5:278-281; Karlsson et al. (1991) J.Immunol. Methods 145:229-240; Malmqvist (1993) Curr. Opin. Immunol.5:282-286; for review, see Hoogenboom, Overview of AntibodyPhage-Display Technology and Its Applications, from Methods in MolecularBiology: Antibody Phage Display: Methods and Protocols (2002) 178:1-37at 19 (O'Brien and Aitken, eds., Human Press, Totowa, N.J.).

In certain embodiments, the binding kinetics or binding affinity of aFab fragment that specifically binds to ANGPTL3 is determined. Incertain instances, Fab fragments have the property of not multimerizing.Multimerization can, in certain instances, complicate the measurement ofbinding kinetics and binding affinity in “solid phase” methods. See,e.g., Hoogenboom, Overview of Antibody Phage-Display Technology and ItsApplications, from Methods in Molecular Biology: Antibody Phase Display:Methods and Protocols (2002) 178:1-37 at 19 (O'Brien and Aitken, eds.,Human Press, Totowa, N.J.). Thus, in certain embodiments, a Fab fragmentthat specifically binds to ANGPTL3 is suitable for use in a bindingassay in which antigen is immobilized to a solid support, such as, forexample, an ELISA-based assay or a Biacore assay. In certainembodiments, Fab fragments are generated from an intact antibody thatspecifically binds to ANGPTL3 using enzymatic methods. In certainembodiments, Fab fragments are produced by expressing nucleic acidsencoding Fab fragments in a recombinant expression system, such as thosedescribed above, Part V.D.3.

In certain embodiments, the binding kinetics or binding affinity of anantibody against ANGPTL3 is determined using “solution phase” methods.In such methods, the kinetics or affinity of binding is measured for anantibody-antigen complex in solution. Certain such methods are known tothose skilled in the art. A nonlimiting example of such a method is the“kinetic exclusion assay,” or “KinExA.” See, e.g., Blake et al. (1996)J. Biol. Chem. 271:27677-27685; Drake et al. (2004) Anal. Biochem.328:35-43 (comparing Biacore “solid phase” and KinExA “solution phase”methods). In certain embodiments, instrumentation for performing KinExAis supplied by Sapidyne Instruments, Inc. (Boise, Id.).

In certain embodiments, the binding kinetics or binding affinity of amultivalent antibody or an antibody that multimerizes is determinedusing a solution phase method. In certain instances, the measurement ofthe binding kinetics or the binding affinity of a multivalent antibodyor an antibody that multimerizes is amenable to solution phase analysis.

In certain embodiments, the binding affinity of an anti-ANGPTL3antibody, as measured by its K_(D), is about 10⁻⁶M or less. In certainembodiments, the binding affinity of an anti-ANGPTL3 antibody is about10⁻⁷ M, about 10⁻⁸ M, or about 10⁻⁹ M or less. In certain suchembodiments, an anti-ANGPTL3 antibody may be used as a therapeuticantibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134. Incertain embodiments, binding affinities of less than 10⁻⁹ M (e.g.,binding affinities from about 500 pM to about 0.5 pM, including but notlimited to, binding affinities from about 100 pM to about 5 pM) areachievable, e.g., using affinity maturation techniques. See, e.g., Boderet al. (2000) Proc. Nat'l Acad. Sci. USA 97:10701-10705. In certainembodiments, the binding affinity of an anti-ANGPTL3 antibody is lessthan about 5×10⁻⁸ M.

In certain embodiments, a monoclonal antibody that was raised againstmouse ANGPTL3 is screened for specific binding to human ANGPTL3 usingcertain routine detection methods, e.g., such as those described herein.The ability of a monoclonal antibody to bind both mouse and humanANGPTL3 (i.e., to demonstrate “cross-reactivity”) indicates the presenceof the same epitope in mouse and human ANGPTL3. In certain embodimentsof detection methods that use denaturing conditions (e.g., Westernblot), cross-reactivity indicates that a mouse monoclonal antibody bindsto the same “linear” epitope in mouse and human ANGPTL3. In certainembodiments of detection methods that use non-denaturing conditions,cross-reactivity indicates that a mouse monoclonal antibody binds to thesame epitope (e.g., a linear epitope or a conformational epitope) inmouse and human ANGPTL3.

2. Certain Methods for Epitope Mapping

In various embodiments, the epitope to which a monoclonal antibody bindsis identified by any of a number of assays. Certain exemplary assays aredescribed, for example, in Morris, Methods in Molecular Biology Vol. 66:Epitope Mapping Protocols (1996) (Humana Press, Totowa, N.J.). Forexample, epitope mapping may be achieved by gene fragment expressionassays or peptide-based assays. In certain embodiments of a genefragment expression assay, for example, nucleic acids encoding fragmentsof ANGPTL3 are expressed in prokaryotic cells and isolated. In certainsuch embodiments, the ability of a monoclonal antibody to bind thosefragments is then assessed, e.g., by immunoprecipitation orimmunoblotting. In certain embodiments, nucleic acids encoding fragmentsof ANGPTL3 are transcribed and translated in vitro in the presence ofradioactive amino acids. The radioactively labeled fragments of ANGPTL3are then tested for binding to a monoclonal antibody. In certainembodiments, fragments of ANGPTL3 are generated by proteolyticfragmentation. In certain embodiments, an epitope is identified usinglibraries of random peptides displayed on the surface of phage or yeast.In certain embodiments, an epitope is identified by testing a library ofoverlapping synthetic peptide fragments of ANGPTL3 for binding to amonoclonal antibody. In certain embodiments, an epitope is identifiedusing a competition assay, such as those described below. In certainembodiments, an epitope may be further defined using alanine-scanningmutagenesis, e.g., as described below.

3. Certain Competition Assays

In certain embodiments, monoclonal antibodies that bind to the sameepitope of ANGPTL3 as a monoclonal antibody of interest are identified.In certain embodiments, such monoclonal antibodies are identified byepitope mapping, e.g., as described above. In certain embodiments, suchmonoclonal antibodies are identified by routine competition assays. See,e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). In anonlimiting exemplary competition assay, ANGPTL3 or a fragment thereofis immobilized onto the wells of a multiwell plate. In certain suchembodiments, the monoclonal antibody of interest is labeled with afluorescent label (in certain embodiments, fluorescein isothiocyanate)by standard methods. In certain such embodiments, mixtures of thelabeled monoclonal antibody of interest and an unlabeled test monoclonalantibody are added to the wells. In certain such embodiments, thefluorescence in each well is quantified to determine the extent to whichthe unlabeled test monoclonal antibody blocks the binding of the labeledmonoclonal antibody of interest. In certain embodiments, monoclonalantibodies are deemed to share an epitope if each blocks the binding ofthe other by 50% or greater. Exemplary competition assays are alsodescribed, e.g., in Morris, Methods in Molecular Biology Vol. 66:Epitope Mapping Protocols (1996) (Humana Press, Totowa, N.J.). Anonlimiting exemplary competition assay is provided below, Part VI.O.

4. Certain Assays for Identifying Neutralizing Antibodies

In certain embodiments, monoclonal antibodies are screened for thosethat are neutralizing antibodies, i.e., those that reduce an activity ofANGPTL3 in vivo and/or in vitro. In certain embodiments, an activity ofANGPTL3 is the ability of ANGPTL3 to inhibit LPL. Thus, in certainembodiments, a neutralizing antibody is identified by its ability toincrease LPL activity in the presence of ANGPTL3. In certain suchembodiments, a neutralizing antibody increases LPL activity by at leastabout 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, relative to acontrol antibody. Certain exemplary assays for measuring LPL activity invivo and in vitro are known in the art.

In certain embodiments, a neutralizing antibody that reduces an activityof ANGPTL3 in vivo is identified by its ability to decrease the level ofat least one serum lipid. Certain exemplary serum lipids include, butare not limited to, triglycerides, cholesterol, and free fatty acids. Incertain such embodiments, a neutralizing antibody decreases the level ofat least one serum lipid by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95%, relative to a control antibody. In certainembodiments, a neutralizing antibody that reduces an activity of ANGPTL3in vivo is identified by its ability to counteract or confer protectionfrom certain effects of a fat-containing diet. Certain exemplary effectsinclude, but are not limited to, weight gain, obesity, glucoseintolerance (hyperglycemia), insulin insensitivity (hyerpinsulinemia),hepatic steatosis (fatty liver), and intramyocellular lipidaccumulation.

G. Certain Pharmaceutical Compositions and Methods of Treatment UsingNeutralizing Monoclonal Antibodies

In certain embodiments, a neutralizing antibody may be used as atherapeutic antibody. Certain exemplary neutralizing antibodies to beused as therapeutic antibodies include, but are not limited to, chimericantibodies, humanized antibodies, and human antibodies. Those skilled inthe art are familiar with the use of certain antibodies as therapeuticagents. For example, over a dozen antibodies have been approved by theFDA for use as therapeutic agents since the mid-1980s. See, e.g., Hudsonet al. (2003) Nat. Med. 9:129-134; Gura (2002) Nature 417:584-586;Brekke et al. (2002) Nat. Reviews 2:52-62. Certain FDA-approvedantibodies include those used to treat various cancers, inflammation,and viral infections and to prevent transplant rejection. See, e.g.,Gura (2002) Nature 417:584-586; Brekke et al. (2002) Nat. Reviews2:52-62. Furthermore, over a dozen antibodies are currently in clinicaltrials. See, e.g., Brekke et al. (2002) Nat. Reviews 2:52-62.

In certain embodiments, methods are provided for treating a disorder oflipid metabolism comprising administering an effective amount of aneutralizing antibody against ANGPTL3. In certain embodiments, methodsare provided for treating an acute disorder of lipid metabolismcomprising administering an effective amount of a neutralizing antibodyagainst ANGPTL3. In certain embodiments, methods are provided fortreating a chronic disorder of lipid metabolism comprising administeringan effective amount of a neutralizing antibody against ANGPTL3. Incertain embodiments, a method for treating a disorder of lipidmetabolism further comprises administering an effective amount of aneutralizing antibody against ANGPTL4. See, e.g., PCT Publication No. WO2006/074228.

As used herein, “disorders of lipid metabolism” include, but are notlimited to, disorders that can lead to secondary hyperlipidemia(including hypertriglyceridemia and hypercholesterolemia). Certainexemplary disorders of lipid metabolism include, but are not limited to,atherosclerosis, dyslipidemia, hypertriglyceridemia (includingdrug-induced hypertriglyceridemia, diuretic-inducedhypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergicblocking agent-induced hypertriglyceridemia, estrogen-inducedhypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia,retinoid-induced hypertriglyceridemia, cimetidine-inducedhypertriglyceridemia, and familial hypertriglyceridemia), acutepancreatitis associated with hypertriglyceridemia, chylomicron syndrom,chylomicronemia, Apo-E deficiency, LPL deficiency or hypoactivity,hyperlipidemia (including familial combined hyperlipidemia),hypercholesterolemia, gout associated with hypercholesterolemia,xanthomatosis (subcutaneous cholesterol deposits), coronary arterydisease (also called ischaemic heart disease), inflammation associatedwith coronary artery disease, restenosis, peripheral vascular diseases,and stroke. Certain exemplary disorders of lipid metabolism include, butare not limited to, disorders related to body weight, such as obesity,metabolic syndrome including independent components of metabolicsyndrome (e.g., central obesity, FBG/pre-diabetes/diabetes,hypercholesterolemia, hypertriglyceridemia, and hypertension),hypothyroidism, uremia, and other conditions associated with weight gain(including rapid weight gain), weight loss, maintenance of weight loss,or risk of weight regain following weight loss. Certain exemplarydisorders of lipid metabolism include, but are not limited to, relatedblood sugar disorders, such as diabetes, hypertension, and polycysticovarian syndrome related to insulin resistance. Certain exemplarydisorders of lipid metabolism include, but are not limited to, renaltransplantation, nephrotic syndrome, Cushing's syndrome, acromegaly,systemic lupus erythematosus, dysglobulinemia, lipodystrophy,glycogenosis type I, and Addison's disease.

Disorders of lipid metabolism include, but are not limited to secondaryhypertriglycerolemia (HTG, including but not limited to types I, V, andIV), including but not limited to, HTG due to diet (including, but notlimited to, excessive alcohol consumption, weight gain, and obesity),drugs (including but not limited to, exogenous estrogen, tamoxifen,retinoids, thiazides, chlorthalidone, beta-clockers, protease inhibitors(including but not limited to ritonavir), propofol infusion, andparenteral lipid infusions), disorders of metabolism (including but notlimited to diabetes, pregnancy, chronic renal failure, hypothyroidism,familial hyperlipidemia, and pancreatitis).

Disorders of lipid metabolism include, but are not limited to, lipiddisorders associated with vascular access dysfunction, lipid disordersassociated with proliferative diseases, including but not limited to,neoplasia (including but not limited to prostate, kidney, liver, breast,ovarian, lung, and pancreatic cancers), disorders that occur in responseto inflammation, including but not limited to, those associated with,e.g., infectious diseases, wound healing, immunodeficiency syndromes(AIDS and others, including but not limited to those syndromesassociated with aberrant development), scar formation, atherosclerosis,restenosis and transplantation rejection, autoimmune disorders, andchronic inflammatory diseases and disorders, which include but are notlimited to, diseases including but not limited to rheumatoid arthritis,systemic lupus erythromatosis, and disorders including but not limitedto Crohn's disease, colitis, inflammatory bowel disease, reactivearthritis, including Lyme disease, insulin dependent diabetes, organspecific autoimmunity, multiple sclerosis, Hashimoto's thyroiditis andGrave's disease, Sjogren's syndrome, contact dermatitis, psoriasis,scleroderma, graft versus host disease, sarcoidosis, malaria, sepsis,pancreatitis, atopic conditions, including but not limited to asthma andallergy, including but not limited to allergic rhinitis,gastrointestinal allergies, including but not limited to food allergies,eosinophilia, conjunctivitis and glomerular nephritis, blood coagulationdisorders, endotoxic shock and other inflammation mediated disorderssuch as sleep apnea and sleepiness.

In certain embodiments, methods are provided for treating a disorder oflipid metabolism comprising administering an effective amount of anantibody to ANGPTL3 and at least one additional therapeutic agent. Incertain such embodiments, an additional therapeutic agent isadministered in an effective amount. In certain embodiments, anadditional therapeutic agent is another antibody to ANGPTL3. In certainembodiments, an additional therapeutic is a neutralizing antibodyagainst ANGPTL4. See, e.g., PCT Publication No. WO 2006/074228. Incertain embodiments, an additional therapeutic agent is a non-antibodyagent. In certain embodiments, an additional therapeutic agent is anagent that lowers the level of one or more serum lipids. Certainexemplary additional therapeutic agents include, but are not limited to,cholesterol synthesis inhibitors (statins), such as HMG-CoA reductaseinhibitors (e.g., lovastatin, simvastatin, pravastatin, andfluvastatin); bile sequestering agents, such as cholestyramine and otherresins; VLDL secretion inhibitors, such as niacin; lipophilicantioxidants, such as Probucol; acyl-CoA cholesterol acyl transferaseinhibitors; farnesoid X receptor antagonists; sterol regulatory bindingprotein cleavage activating protein (SCAP) activators; microsomaltriglyceride transfer protein (MTP) inhibitors; and ApoE-relatedpeptide. In certain embodiments, the additional therapeutic agent is anagent that raises high density lipoprotein (HDL). Nonlimiting examplesof such agents include, but are not limited to, cholesteryl estertransfer protein (CETP) inhibitors.

In certain embodiments, a pharmaceutical composition is provided thatcomprises an effective amount of an antibody to ANGPTL3 and apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant. In certain embodiments, a pharmaceuticalcomposition is provided that comprises an effective amount of anantibody to ANGPTL3 and an effective amount of at least one additionaltherapeutic agent, together with a pharmaceutically acceptable diluent,carrier, solubilizer, emulsifier, preservative and/or adjuvant. Incertain embodiments, the at least one additional therapeutic agent isselected from those described above.

In certain embodiments, formulation materials for pharmaceuticalcompositions are nontoxic to recipients at the dosages andconcentrations employed.

In certain embodiments, the pharmaceutical composition comprisesformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In certain embodiments, suitableformulation materials include, but are not limited to, amino acids (forexample, glycine, glutamine, asparagine, arginine and lysine);antimicrobials; antioxidants (for example, ascorbic acid, sodium sulfiteand sodium hydrogen-sulfite); buffers (for example, borate, bicarbonate,Tris-HCl, citrates, phosphates and other organic acids); bulking agents(for example, mannitol and glycine); chelating agents (for example,ethylenediamine tetraacetic acid (EDTA)); complexing agents (forexample, caffeine, polyvinylpyrrolidone, beta-cyclodextrin, andhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides,disaccharides, and other carbohydrates (for example, glucose, mannoseand dextrins); proteins (for example, serum albumin, gelatin andimmunoglobulins); coloring, flavoring, and diluting agents; emulsifyingagents; hydrophilic polymers (for example, polyvinylpyrrolidone); lowmolecular weight polypeptides; salt-forming counterions (for example,sodium); preservatives (for example, benzalkonium chloride, benzoicacid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid and hydrogen peroxide);solvents (for example, glycerin, propylene glycol and polyethyleneglycol); sugar alcohols (for example, mannitol and sorbitol); suspendingagents; surfactants or wetting agents (for example, pluronics, PEG,sorbitan esters, polysorbates (for example, polysorbate 20 andpolysorbate 80), triton, tromethamine, lecithin, cholesterol, andtyloxapal); stability enhancing agents (for example, sucrose andsorbitol); tonicity enhancing agents (for example, alkali metal halides(for example, sodium or potassium chloride), mannitol, and sorbitol);delivery vehicles; diluents; excipients; and pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18^(th) Edition, A. R. Gennaro,ed., Mack Publishing Company (1990).

In certain embodiments, an antibody to ANGPTL3 or other therapeuticmolecule is linked to a half-life extending vehicle. Certain exemplaryhalf-life extending vehicles are known in the art. Certain such vehiclesinclude, but are not limited to, the Fc domain, polyethylene glycol, anddextran. Certain such vehicles are described, e.g., in published PCTApplication No. WO 99/25044.

In certain embodiments, an optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format, and desired dosage.See, e.g., Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release, or rate of in vivo clearance of aneutralizing antibody.

In certain embodiments, a primary vehicle or carrier in a pharmaceuticalcomposition may be either aqueous or non-aqueous in nature. For example,in certain embodiments, a suitable vehicle or carrier may be water forinjection, physiological saline solution, or artificial cerebrospinalfluid, possibly supplemented with other materials common in compositionsfor parenteral administration. Certain exemplary vehicles include, butare not limited to, neutral buffered saline and saline mixed with serumalbumin. In certain embodiments, pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,which may further include sorbitol or a suitable substitute therefor. Incertain embodiments, a composition comprising an antibody to ANGPTL3,with or without at least one additional therapeutic agents, may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. In certain embodiments, a composition comprising anantibody to ANGPTL3, with or without at least one additional therapeuticagent, may be formulated as a lyophilizate using appropriate excipientssuch as sucrose.

In certain embodiments, a pharmaceutical composition is selected forparenteral delivery. In certain embodiments, a pharmaceuticalcomposition is selected for inhalation or for delivery through thedigestive tract, such as orally. Certain exemplary techniques forpreparing pharmaceutically acceptable compositions are within the skillof one skilled in the art.

In certain embodiments, formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a pharmaceutical composition may be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising the desired antibodyto ANGPTL3, with or without additional therapeutic agents, in apharmaceutically acceptable vehicle. In certain embodiments, a vehiclefor parenteral injection is sterile distilled water in which theantibody to ANGPTL3, with or without at least one additional therapeuticagent, is formulated as a sterile, isotonic solution, properlypreserved. In certain embodiments, the preparation can involve theformulation of the desired molecule with an agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (such aspolylactic acid or polyglycolic acid), beads or liposomes, that mayprovide for the controlled or sustained release of the product which maythen be delivered via a depot injection. In certain embodiments,hyaluronic acid may also be used, and may have the effect of promotingsustained duration in the circulation. In certain embodiments,implantable drug delivery devices may be used to introduce the desiredmolecule.

In certain embodiments, a pharmaceutical composition may be formulatedfor inhalation. In certain embodiments, an antibody to ANGPTL3, with orwithout at least one additional therapeutic agent, may be formulated asa dry powder for inhalation. In certain embodiments, an inhalationsolution comprising an antibody to ANGPTL3, with or without at least oneadditional therapeutic agent, may be formulated with a propellant foraerosol delivery. In certain embodiments, solutions may be nebulized.

In certain embodiments, a formulation may be administered orally. Incertain embodiments, an antibody to ANGPTL3, with or without at leastone additional therapeutic agent, that is administered in this fashionmay be formulated with or without carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Incertain embodiments, a capsule may be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. In certain embodiments, at least one additional agent can beincluded to facilitate absorption of the antibody to ANGPTL3 with orwithout any additional therapeutic agents. In certain embodiments,diluents, flavorings, low melting point waxes, vegetable oils,lubricants, suspending agents, tablet disintegrating agents, and/orbinders may also be employed.

In certain embodiments, a pharmaceutical composition comprises aneffective amount of an antibody to ANGPTL3, with or without at least oneadditional therapeutic agent, in a mixture with non-toxic excipientswhich are suitable for the manufacture of tablets. In certainembodiments, by dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions may be prepared in unit-dose form.Certain exemplary excipients include, but are not limited to, inertdiluents (for example, calcium carbonate, sodium carbonate, sodiumbicarbonate, lactose, and calcium phosphate); binding agents (forexample, starch, gelatin, and acacia); and lubricating agents (forexample, magnesium stearate, stearic acid, and talc).

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations comprising an antibody to ANGPTL3,with or without at least one additional therapeutic agent, in sustained-or controlled-delivery formulations. Certain exemplary sustained- orcontrolled-delivery formulations include, but are not limited to,liposome carriers, bio-erodible microparticles, porous beads, and depotinjections. Certain exemplary techniques for preparing certainformulations are known to those skilled in the art. In certainembodiments, sustained-release preparations may include semipermeablepolymer matrices in the form of shaped articles, e.g. films ormicrocapsules. Certain exemplary sustained release matrices include, butare not limited to, polyesters, hydrogels, polylactides (see, e.g., U.S.Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid andgamma ethyl-L-glutamate (see, e.g., Sidman et al. (1983) Biopolymers22:547-556), poly (2-hydroxyethyl-methacrylate) (see, e.g., Langer etal. (1981) J. Biomed. Mater. Res. 15:167-277 and Langer (1982) Chem.Tech. 12:98-105), ethylene vinyl acetate (Langer et al., supra), andpoly-D(−)-3-hydroxybutyric acid (EP 133,988). In certain embodiments,sustained release compositions may include liposomes, which can beprepared, in certain embodiments, by any of several methods known in theart. See e.g., Eppstein et al. (1985) Proc. Natl. Acad. Sci. USA,82:3688-3692; EP 036,676; EP 088,046; and EP 143,949.

In certain embodiments, a pharmaceutical composition to be used for invivo administration typically is sterile. In certain embodiments, thismay be accomplished by filtration through sterile filtration membranes.In certain embodiments, where the composition is lyophilized,sterilization using this method may be conducted either prior to orfollowing lyophilization and reconstitution. In certain embodiments, thecomposition for parenteral administration may be stored in lyophilizedform or in a solution. In certain embodiments, parenteral compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it may be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations may be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits for producing a single-dose administrationunit are provided. In certain embodiments, the kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. In certain embodiments, kits containingsingle or multi-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising an antibody to ANGPTL3, with or without at leastone additional therapeutic agent, to be employed therapeutically willdepend, for example, upon the context and objectives of treatment. Oneskilled in the art will appreciate that the appropriate dosage levelsfor treatment, according to certain embodiments, will thus varydepending, in part, upon the molecule delivered, the indication forwhich the antibody to ANGPTL3, with or without at least one additionaltherapeutic agent, is being used, the route of administration, and thesize (body weight, body surface or organ size) and/or condition (the ageand general health) of the patient. In certain embodiments, theclinician may titer the dosage and modify the route of administration toobtain the optimal therapeutic effect. In certain embodiments, a typicaldosage may range from about 0.1 μg/kg of patient body weight, up toabout 100 mg/kg or more, depending on the factors mentioned above. Incertain embodiments, the dosage may range from 0.1 μg/kg up to about 100mg/kg; 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg,including all points (including fractions) between any of the foregoingendpoints. In certain embodiments, the dosage is between about 10 mg/kgbody weight and about 60 mg/kg body weight. In certain embodiments, thedosage is about 10 mg/kg body weight, about 20 mg/kg body weight, about30 mg/kg body weight, about 40 mg/kg body weight, about 50 mg/kg bodyweight, or about 60 mg/kg body weight.

In certain embodiments, a human dose of a neutralizing antibody againstANGPTL3 is determined based on the efficacious dose of the same antibodyin mice. In certain embodiments, a human dose of a neutralizing antibodyagainst ANGPTL3 is determined using “Guidance for Industry: Estimatingthe Maximum Safe Starting Dose in Initial Clinical Trials forTherapeutics in Adult Healthy Volunteers,” U.S. Department of Health andHuman Services, Food and Drug Administration, and Center for DrugEvaluation and Research (CDER), July 2005 (Pharmacology and Toxicology).In certain embodiments, a human dose of a neutralizing antibody againstANGPTL3 is between 0.07 mg/kg and 7 mg/kg. In certain embodiments, ahuman dose of a neutralizing antibody against ANGPTL3 is between 0.1mg/kg and 5 mg/kg. In certain embodiments, a human dose of aneutralizing antibody against ANGPTL3 is between 0.1 mg/kg and 2 mg/kg.In various embodiments, a neutralizing antibody against ANGPTL3 isadministered to a patient twice per week, once per week, once every twoweeks, or once per month.

In certain embodiments, a suitable dosage may be determined by oneskilled in the art, for example, based on animal studies.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of an antibody to ANGPTL3 and, ifapplicable, any additional therapeutic agents in the formulation used.In certain embodiments, a clinician will administer the compositionuntil a dosage is reached that achieves the desired effect. In certainembodiments, the composition may therefore be administered as a singledose, or as two or more doses (which may or may not contain the sameamount of the desired molecule) over time, or as a continuous infusionvia an implantation device or catheter. In certain embodiments, furtherrefinement of the appropriate dosage is routinely made by those skilledin the art and is within the ambit of tasks routinely performed by them.In certain embodiments, appropriate dosages may be ascertained throughuse of appropriate dose-response data. In certain embodiments, a patientreceives one dose of a pharmaceutical composition comprising an antibodyto ANGPTL3. In certain embodiments, a patient receives one, two, three,or four doses per day of a pharmaceutical composition comprising anantibody to ANGPTL3. In certain embodiments, a patient receives one,two, three, four, five, or six doses per week of a pharmaceuticalcomposition comprising an antibody to ANGPTL3. In certain embodiments, apatient receives one or two doses per month of a pharmaceuticalcomposition comprising an antibody to ANGPTL3.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,intra-ocular, intraarterial, intraportal, or intralesional routes; bysustained release systems or by implantation devices. In certainembodiments, the compositions may be administered by bolus injection orcontinuously by infusion, or by implantation device.

In certain embodiments, the composition may be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

In certain embodiments, it may be desirable to use a pharmaceuticalcomposition comprising an antibody to ANGPTL3, with or without at leastone additional therapeutic agent, in an ex vivo manner. In certain suchinstances, cells, tissues and/or organs that have been removed from thepatient are exposed to a pharmaceutical composition comprising anantibody to ANGPTL3, with or without at least one additional therapeuticagent, after which the cells, tissues and/or organs are subsequentlyimplanted back into the patient.

In certain embodiments, an antibody to ANGPTL3, with or without at leastone additional therapeutic agent, is delivered by implanting certaincells that have been genetically engineered, using methods such as thosedescribed herein, to express and secrete the polypeptides. In certainembodiments, such cells may be animal or human cells, and may beautologous, heterologous, or xenogeneic. In certain embodiments, thecells may be immortalized. In certain embodiments, in order to decreasethe chance of an immunological response, the cells may be encapsulatedto avoid infiltration of surrounding tissues. In certain embodiments,the encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

H. Certain Methods of Detection and Diagnosis

In certain embodiments, antibodies against ANGPTL3 are used to detectthe presence of ANGPTL3 in vivo or in vitro. In certain embodiments, thelevel of ANGPTL3 in vivo is correlated with a medical condition, such asa disorder of lipid metabolism, thereby allowing diagnosis of themedical condition. Certain exemplary medical conditions that may bediagnosed by an antibody against ANGPTL3 are set forth above.

Certain exemplary detection methods are known in the art and include,but are not limited to, ELISA, radioimmunoassay, immunoblot, Westernblot, immunofluorescence, and immunoprecipitation. In certainembodiments, antibodies against ANGPTL3 are modified so that they may bedirectly detected, for example, by linking the antibody to a label.Certain exemplary labels include, but are not limited to, fluorophores,chromophores, radioactive atoms, electron-dense reagents, enzymes, andligands. In certain embodiments, antibodies against ANGPTL3 are detectedby using a labeled “secondary” antibody that binds to a class ofantibodies (e.g., a goat anti-mouse antibody).

I. Certain Screening Methods for ANGPTL3 Antagonists and Agonists

In certain embodiments, a method of screening for an agent that binds toANGPTL3 is provided. In certain embodiments, a screening methodcomprises exposing ANGPTL3 to one or more candidate agents undersuitable conditions and assessing binding of ANGPTL3 to the one or morecandidate agents. In certain embodiments, a screening method comprisesusing an antibody against ANGPTL3 in a competitive binding assay. Incertain such embodiments, a first binding mixture comprising an antibodyagainst ANGPTL3 and ANGPTL3 is used. The amount of binding betweenANGPTL3 and the antibody in the first binding mixture (M₀) is measured.A second binding mixture comprising the antibody, ANGPTL3, and an agentto be screened is also used. The amount of binding between ANGPTL3 andthe antibody in the second binding mixture (M₁) is measured. The amountof binding in the first binding mixture is compared with the amount ofbinding in the second binding mixture, for example, by calculating theM₁/M₀ ratio. An agent is considered to be capable of binding ANGPTL3 ifthe amount of binding of antibody to ANGPTL3 in the second bindingmixture is less than the amount of binding of antibody to ANGPTL3 in thefirst binding mixture. In certain embodiments, an agent that bindsANGPTL3 decreases the binding of antibody to ANGPTL3 by at least about10% (i.e., M₁/M₀<0.9), by at least about 30% (i.e., M₁/M₀<0.7), by atleast about 50% (i.e., M₁/M₀<0.5), by at least about 70% (i.e.,M₁/M₀<0.3), by at least about 80% (i.e., M₁/M₀<0.2), by at least about90% (i.e., M₁/M₀<0.1), or by at least about 95% (i.e., M₁/M₀<0.05).

In certain embodiments, the ANGPTL3 to be used in any of the screeningmethods described above is the N-terminal coiled-coil domain of ANGPTL3or a fragment thereof. Based on the applicants' observation that certainantibodies that bind within the N-terminal coiled-coil domain of ANGPTL3can decrease serum triglyceride and serum cholesterol levels in vivo, anagent (e.g., an antibody or a non-antibody agent) identified by ascreening method as binding to the N-terminal coiled-coil domain ofANGPTL3 is a candidate antagonist of ANGPTL3 activity. In certainembodiments, an agent (e.g., an antibody or a non-antibody agent)identified by a screening method as binding to the SP1 region within theN-terminal coiled-coil domain of ANGPTL3 is a candidate antagonist ofANGPTL3 activity.

In certain embodiments, antagonist activity is verified by demonstratingthat the candidate antagonist neutralizes ANGPTL3 activity in an in vivoor in vitro assay. Certain exemplary assays are described herein. Oneskilled in the art can select and/or adapt an appropriate assay fromthose described herein and/or those known in the art. In certainembodiments, antagonists of ANGPTL3 are used in the treatment ofdisorders of lipid metabolism.

In certain embodiments, methods of screening for agents that bind to thefibrinogen domain of ANGPTL3 are provided. In certain embodiments, anagent that binds within the fibrinogen domain of ANGPTL3 may enhanceANGPTL3 activity. Thus, an agent (e.g., an antibody or a non-antibodyagent) identified by a screening method as binding to the fibrinogendomain of ANGPTL3 is a candidate agonist of ANGPTL3 activity. In certainembodiments, agonist activity is verified by demonstrating that thecandidate agonist enhances ANGPTL3 activity in an in vitro or an in vivoassay. One skilled in the art can select and/or adapt an appropriateassay based on the assays known in the art and/or the assays describedherein. In certain embodiments, agonists of ANGPTL3 are used in thetreatment of certain disorders related to excessive weight loss, such asanorexia nervosa, bulimia nervosa and the cachexia (wasting) associatedwith diseases such as cancer, cystic fibrosis, and AIDS.

Certain exemplary agents that can be screened for binding to ANGPTL3include, but are not limited to, antibodies, small molecules (e.g.,organic compounds, organometallic compounds, salts of organic andorganometallic compounds, saccharides, amino acids, nucleosides, andnucleotides), aptamers, peptides, and peptide mimetics. Certainexemplary peptides include soluble peptides, which include, but are notlimited to, members of random peptide libraries (see, e.g., Lam et al.(1991) Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) andmembers of combinatorial chemistry-derived molecular libraries made ofD- and/or L-configuration amino acids; and phosphopeptides, whichinclude, but are not limited to, members of random or partiallydegenerate, directed phosphopeptide libraries (see, e.g., Songyang(1993) Cell 72:767-778).

In certain embodiments, computer modeling and searching technologiespermit identification of compounds, or the improvement of alreadyidentified compounds, that bind ANGPTL3. Certain exemplary molecularmodeling systems include, but are not limited to, the CHARMM and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMM performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

J. Nucleic Acid Antagonists of ANGPTL3

In certain embodiments, an isolated nucleic acid that decreases theexpression of a nucleic acid encoding ANGPTL3 is provided. In certainembodiments, the nucleic acid encoding ANGPTL3 encodes mouse ANGPTL3. Incertain embodiments, the nucleic encoding ANGPTL3 encodes human ANGPTL3.In certain embodiments, the isolated nucleic acid is an antisensenucleic acid. In certain such embodiments, the antisense nucleic acid isa single stranded DNA molecule that promotes the degradation of a targetmRNA by an RNaseH-based mechanism. In certain embodiments, an antisensenucleic acid is an oligonucleotide of about 8-30 nucleotides in length(including all points between the end points). In certain embodiments,an antisense nucleic acid is an oligonucleotide of about 18-26nucleotides in length.

In certain embodiments, an antisense nucleic acid encompasses an RNAmolecule that reduces expression of a target nucleic acid by an RNAinterference (RNAi)-based mechanism. Certain exemplary RNA moleculessuitable for RNAi include, but are not limited to, short interferingRNAs (siRNAs), microRNAs (mRNAs), tiny non-coding RNAs (tncRNAs), andsmall modulatory RNA (smRNA). For review of certain exemplary RNAimechanisms and RNA molecules for use in RNAi, see, e.g., Novina et al.(2004) Nature 430:161-164.

In certain embodiments, an siRNA that decreases expression of a nucleicacid encoding ANGPTL3 is provided. In certain embodiments, an siRNA isan oligonucleotide of about 18-26 nucleotides in length (including allpoints between the endpoints). In certain embodiments, an siRNA is anoligonucleotide of about 20-24 nucleotides in length, or anoligonucleotide of about 21-23 nucleotides in length. In certainembodiments, an siRNA is double-stranded RNA. In certain embodiments, ansiRNA will induce the degradation of a target mRNA molecule that iscomplementary to the antisense strand of the siRNA. See, e.g., Novina etal. (2004)Nature 430:161-164.

The activity of an antisense nucleic acid, such as an antisense DNAmolecule or an siRNA, is often affected by the secondary structure ofthe target mRNA. See, e.g., Vickers et al. (2003) J. Biol. Chem.278:7108-7118. Thus, in certain embodiments, an antisense nucleic acidis selected that is complementary to a region of a target mRNA that isavailable for base-pairing. In certain embodiments, a suitable region ofa target mRNA is identified by performing a “gene walk,” e.g., byempirically testing a number of antisense oligonucleotides for theirability to hybridize to various regions along a target mRNA and/or toreduce target mRNA expression. See, e.g., Vickers et al. (2003) J. Biol.Chem. 278:7108-7118; Hill et al. (1999) Am. J. Respir. Cell Mol. Biol.21:728-737. In certain embodiments, a suitable region of a target mRNAis identified using an mRNA secondary structure prediction program orrelated algorithm to identify regions of a target mRNA that do nothybridize to any other regions of the target mRNA. See, e.g., Hill etal. (1999) Am. J. Respir. Cell Mol. Biol. 21:728-737. In certainembodiments, a combination of both of the above methods is used toidentify a suitable region of a target mRNA. See e.g., Hill et al.(1999) Am. J. Respir. Cell Mol. Biol. 21:728-737.

In certain embodiments, a method of reducing ANGPTL3 activity byreducing expression of a nucleic acid encoding ANGPTL3 is provided. Incertain embodiments, the method comprises reducing expression of anucleic acid encoding ANGPTL3 in a cell in vitro or in vivo. In certainembodiments, the method comprises administering an antisense nucleicacid that reduces expression of a nucleic acid encoding ANGPTL3 to acell in vitro or in vivo. In certain embodiments, the nucleic acidencoding ANGPTL3 encodes human ANGPTL3. In certain embodiments, thenucleic acid encoding ANGPTL3 encodes mouse ANGPTL3.

In certain embodiments, a method of treating a disorder of lipidmetabolism, such as any of those described above, is provided. Incertain embodiments, the method comprises administering to a patient aneffective amount of an antisense nucleic acid that reduces expression ofa nucleic acid encoding ANGPTL3. In certain embodiments, antisensenucleic acid is delivered to an organ that expresses a nucleic acidencoding ANGPTL3.

ANGPTL3 is expressed primarily in the liver. Oike, Y. et al. (2005)TRENDS Mol. Med. 11(10):473-479. In mice, expression is apparently notaffected by short-term fasting. Oike, Y. et al. (2005) TRENDS Mol. Med.11(10):473-479. However, expression is increased with cholesterolfeeding and in certain mouse models of obesity and diabetes (e.g., db/dband ob/ob mice), and mice with streptozotocin-induced type I diabetes),suggesting that ANGPTL3 can contribute to the dyslipidemia of diabetesand the metabolic syndrome. Oike, Y. et al. (2005) TRENDS Mol. Med. 11(10):473-379. Thus, in certain embodiments, antisense nucleic acid isdelivered to the liver. Certain exemplary guidance for the in vivoadministration of antisense nucleic acids and the sustained delivery ofantisense nucleic acids in vivo, including sustained delivery tospecific organs such as the liver, is provided, for example, in Khan etal. (2004) J. Drug Targeting 12:393-404. In certain embodiments,sustained delivery is achieved by administering antisense nucleic acidthat is encapsulated or otherwise contained by a biodegradable polymer.For example, in certain embodiments, antisense nucleic acid may becontained within poly(glycolic acid) (PLGA) microspheres (e.g., 0.5-20μm; 3000 MW). In certain embodiments, the antisense nucleic acid isconjugated to a lipophilic moiety. See Khan et al. (2004) J. DrugTargeting 12:393-404.

VI. EXAMPLES A. Mouse Care and Dietary Studies

Mouse studies were performed according to federal guidelines. Mice werehoused at 24° C. on a fixed 12 hour light/2 hour dark cycle and had adlibitum access to water and rodent chow (22% calories from fat) (productno. 5021; Purina, St. Louis, Mo.) as indicated below. Mice referred tobelow as being in the “fasted state” were deprived of food for 16 hours.

B. In Vivo Overexpression of Human ANGPTL3 in Mice

cDNA encoding full-length human ANGPTL3 (SEQ ID NO: 6) was inserted intothe Ad E1-deleted region of the adenovirus vector pFAD, thereby placingthe cDNA under the control of the cytomegalovirus promoter. See Hitt etal., “Construction and propagation of human adenovirus vectors,” in CellBiology: A Laboratory Handbook Vol. 1, pp. 500-512 (J. E. Celis, ed.,2^(nd) ed. 1998). The resultant construct, Ad5-hAngptl3T, was used toinfect CHO cells. Empty Ad5 virus was also used to infect CHO cells as acontrol. Expression of human ANGPTL3 was confirmed by Western blot ofinfected CHO cell extracts.

Ad5-hAngptl3T was injected into C57BL/6J mice via the tail vein at thefollowing dosages: 2×10⁹ vp, 1×10⁹ vp, 5×10⁸ vp, 2×10⁸ vp. Empty Ad5vector was injected into C57BL/6J mice via the tail vein at 2×10⁹ vp asa control. There were five mice in each group. Blood samples fromadenovirus-infected mice were collected after four days, during whichtime the mice were fed normally (not fasted). Triglyceride levels inserum were measured using the Cobas Integra 500 (Roche, Basel,Switzerland).

The results are shown in FIG. 8. In that experiment, mice injected with2×10⁹ vp or 1×10⁹ vp of Ad5-hAngptl3T had significantly increased serumtriglyceride levels when compared to mice injected with the empty Ad5vector control.

C. Production and Purification of Mouse and Human ANGPTL3

To express recombinant mouse ANGPTL3, CHO cells were infected with1.5×10¹¹ vp of recombinant adenovirus Ad5-mAngptlT. Ad5-mAngptlTexpresses mouse ANGPTL3 with a His₆ tag at the C-terminus (a glycineresidue is included between ANGPTL3 and the His₆ tag), referred to asmANGPTL3T (SEQ ID NO: 2). The medium was changed to serum-free medium(EX-CELL 325-PF CHO medium, 14335, JRH, Lenexa, Kans.) 16-24 hourslater. The conditioned medium was harvested and then replaced with freshserum-free medium every 24-36 hours for a total of 5 harvests.

Conditioned medium (1 L) was loaded onto a 10-12 ml column ofNickel-Chelating Resin (R801-01, Invitrogen, Carlsbad, Calif.). Thecolumn was washed with 5 column volumes of wash buffer (10 mM imidazole,20 mM Tris pH 7.8, 500 mM NaCl). Bound mANGPTL3T was eluted with elutionbuffer (500 mM imidazole, 20 mM Tris pH 7.8, 500 mM NaCl) and collectedin a series of 1.5 ml fractions. The presence of mANGPTL3T in thecollected fractions was determined by western blot and simply bluestaining. Fractions containing mANGPTL3T were pooled together,aliquoted, and frozen at −70° C.

To express recombinant human ANGPTL3, CHO cells were infected with1.5×10¹¹ vp of recombinant adenovirus Ad5-hAngptlT. Ad5-hAngptlTexpresses human ANGPTL3 with a His₆ tag at the C-terminus (a glycineresidue is included between ANGPTL3 and the His₆ tag), referred to ashANGPTL3T (SEQ ID NO: 4). hANGPTL3T was expressed and purified asdescribed above for mANGPTL3T.

D. Production and Purification of rN′-mANGPTL3T and rN′-hANGPTL3T

A polynucleotide sequence encoding amino acids 17 to 240 of mouseANGPTL3 was cloned into expression vector pET22b(+) (Novagen). Thatvector encodes an N-terminal pelB leader sequence, as well as aC-terminal His tag. The resulting expression vector is calledpET-N′-mANGPTL3T. Following translation of the protein and removal ofall but 11 amino acids of the pelB sequence, N′-mANGPTL3T has thesequence shown in SEQ ID NO: 7. That sequence contains 11 amino acidsfrom the pelB sequence, followed by amino acids 17 to 240 of mouseANGPTL3 (underlined in Table 7), followed by a 2 amino acid linker andthe His₆ tag.

Similarly, a polynucleotide sequence encoding amino acids 20 to 243 ofhuman ANGPTL3 was cloned into expression vector pET22b(+) (Novagen).That vector encodes an N-terminal pelB leader sequence, as well as aC-terminal His tag. The resulting expression vector is calledpET-N′-hANGPTL3T. Following translation of the protein and removal ofall but 11 amino acids of the pelB sequence, N′-hANGPTL3T has thesequence shown in SEQ ID NO: 8. That sequence contains 11 amino acidsfrom the pelB sequence, followed by amino acids 20 to 243 of humanANGPTL3 (underlined in Table 7), followed by a 2 amino acid linker andthe His₆ tag.

N′-mANGPTL3T or N′-hANGPTL3T is expressed and purified from E. coli asfollows. Ten ml of LB containing 50 μg/ml of chloramphenicol and 100μg/ml of carbenicillin is inoculated with one colony of E. colitransformed with pET-N′-mANGPTL3T. The culture is incubated at 37° C.overnight. The 10 ml culture is then transferred to 500 ml of LB withoutantibiotics and incubated at 37° C. until the OD₆₀₀ reaches 0.6 (about 2hours). IPTG is added to a final concentration of 1 mM and the cultureis incubated with shaking at 200 rpm at 30° C. for 4 hours. The cultureis then placed on ice for 5 minutes. The cells are pelleted bycentrifuging at 8000 rpm in a JLA 16.25 rotor for 15 minutes. The pelletis then resuspended in 50 ml of lysis buffer (50 mM Tris, pH 7.5, 0.5 MNaCl, 1% Triton X-100, 1× protease inhibitor cocktail (Roche) and 0.25ml PMSF (0.1 M in isopropanol)). The lysed cells are then centrifuged at9700 rpm in a JA25.5 rotor for 30 minutes. The supernatant is removedand further clarified by centrifuging it at 28,000 rpm in an SW28 rotorfor 30 minutes. Recombinant N′-mANGPTL3T or N′-hANGPTL3T can then bepurified from the clarified supernatant using Probond (Ni)chromatography (Invitrogen).

To purify recombinant N′-mANGPTL3T or N′-hANGPTL3T from the insolublepellet remaining after centrifuging the lysed cells, the pellet iswashed with 30 ml lysis buffer and centrifuged at 9700 rpm in a JA25.5rotor. The wash step is repeated twice, for a total of three washes.Insoluble protein from the pellet is then dissolved in 10 ml ofdenaturing buffer (50 mM Tris, pH 8.0, 6 M Guanadine HCl). The solutionis then centrifuged at 28,000 rpm in a JA25.5 rotor for 30 minutes. Thesupernatant is then loaded onto a 5 ml Probond resin column. The columnis washed with 50 ml of washing buffer (50 mM Tris, pH 8.0, 1 M NaCl, 8M urea, 15 mM imidazole). Recombinant protein is refolded in the columnwith a 50 ml gradient going from washing buffer to renaturing buffer (50mM Tris, pH 8.0, 1 M NaCl, 0.5% Tween 20). The recombinant protein isthen eluted with elution buffer (renaturing buffer with 250 mMimidazole). The fractions containing recombinant protein are collectedand dialyzed against storage buffer (50 mM Tris, pH 8.0, 100 mM NaCl,0.2% Tween 20). The purified N′-mANGPTL3T or N′-hANGPTL3T is aliquotedand stored at −70° C.

E. Production and Purification of SP1 and SP2-KLH

Purified SP1 (SEQ ID NOS:9 and 10) and SP2-KLH (SEQ ID NO:62) werepurchased from Sigma Genosys (The Woodlands, Texas). The sequence of theSP1 region of ANGPTL3 is the same in both mouse and human. SP2-KLH wasmade by adding a cysteine to the COOH-terminus of SP1, then conjugatingKeyhole Limpet Hemocyanin to the peptide via the cysteine using standardmethods known in the art.

F. Generation of Monoclonal Antibodies Against ANGPTL3 in Angptl3Knockout Mice

Monoclonal antibodies that cross-react with both human and mouse ANGPTL3were raised in Angptl3 knockout mice using three cohorts of 5-8 mice,each receiving a different series of antigen injections. Cohort 1 wasprimed with mANGPTL3T (SEQ ID NO: 2), produced and purified as describedabove. Cohort 1 was also boosted four times with mANGPTL3T. The finalboost before harvesting the lymphoid tissue was with mANGPTL3T. Cohort 2was primed with rN′-hANGPTL3T (SEQ ID NO: 8), produced and purified asdescribed above. Cohort 2 was boosted once with rN′-mANGPTL3T (SEQ IDNO: 7), produced and purified as described above, and then three timeswith mANGPTL3T (SEQ ID NO: 2). The final boost before harvesting thelymphoid tissue was with hANGPTL3T (SEQ ID NO: 4). Cohort 3 was primedwith hANGPTL3T (SEQ ID NO: 4), produced and purified as described above,and then boosted once with hANGPTL3T and then boosted twice with thesynthetic peptide SP2-KLH (SEQ ID NO: 62) produced and purified asdescribed above. Two high titer animals from cohort 3 were given a finalboost with SP2-KLH (SEQ ID NO: 62) before harvesting lymphoid tissue.The remaining cohort 3 animals (referred to as cohort 4) were boostedfour additional times with the synthetic peptide SP2-KLH (SEQ ID NO: 62)produced and purified as described above. The final boost for cohort 4before harvesting the lymphoid tissue was with SP2-KLH (SEQ ID NO: 62).

Each mouse was primed and boosted as follows. Each mouse was primed with40 μg of purified antigen in Complete Freund's Adjuvantintraperitoneally. The mice were boosted after two weeks with 30 μg ofpurified antigen in Incomplete Freud's Adjuvant (IFA) intraperitoneally,and then boosted again after another two weeks with 20 μg purifiedantigen in IFA intraperitoneally. Alternatively, chitosan-basedadjuvants can be used. See, e.g., U.S. Pat. Nos. 5,912,000; 5,965,144;and 5,980,912. One week after the second boost, serum titers weremeasured by ELISA, as described below, using mANGPTL3 as theantigen-coated plates. Two weeks after the second boost, the mice wereboosted with 10 μg purified antigen in IFA intraperitoneally. One weekafter the third boost, serum titers were again measured by ELISA asabove. For cohort 4, intraperitoneal boosting was continued until hightiters were achieved. Upon generation of high titers, final boosts weredone using either the priming antigen (cohorts 1 and 2) or the boostingantigen (cohorts 3 and 4). The final boost was given about two and ahalf weeks after the penultimate boost, and was done with 10 μg purifiedantigen intravenously.

Splenocytes were harvested three days after the final boost from theimmunized mice and fused with myeloma cells (NSI) using PEG1500 as afusion agent. The resulting cell fusion products were diluted intohybridoma medium and seeded into 96-well tissue culture plates. After 1day, HAT medium was added to the hybridoma cultures. The medium waschanged every three or four days as necessary.

After ten to fourteen days of selection and culture, hybridomas werescreened by ELISA. The hybridomas from cohort 1 were screened usingmANGPTL3T, hANGPTL3T, and N′-hANGPTL3T. The hybridomas from cohort 2were screened using mANGPTL3T and hANGPTL3T. The hybridomas from cohorts3 and 4 were screened using hANGPTL3T and synthetic peptide representingthe SP1 region of ANGPTL3.

ELISAs were performed as described below.

G. ELISA Methods

Antibodies were screened for binding to antigen using ELISA. Antibodiesfrom each cohort were screened for binding to mANGPTL3T, hANGPTL3T,N′-hANGPTL3T, and/or hANGPTL3 SP1 peptide. Ninety-six well NuncMaxi-Sorp ImmunoPlates™ (Nunc #446612, Roskilde, Denmark) were coated byadding 50 μl per well of a 2.5 μg/ml solution of mANGPTL3T, hANGPTL3T,N′-hANGPTL3T, and/or hANGPTL3 SP1 peptide in coating buffer (BupH™Carbonate-Bicarbonate Buffer, Pierce #28382, Rockford, Ill.) overnightat 4° C. Coating buffer was removed and the plate was blocked by adding250 μl per well of blocking buffer (1% Blocker™ BSA, Pierce #37525, inPBS) for two hours at room temperature. 50 μl of hybridoma supernatant(undiluted or diluted in blocking buffer) or isolated anti-ANGPTL3antibody (undiluted or diluted in blocking buffer) were added to thewells and incubated for at least one hour at room temperature. Wellswere washed four times with PBS/Tween 20. 100 μl of diluted (1:5,000 to1:10,000) HRP-conjugated goat anti-mouse IgG (Pierce #31446) were addedto the wells and incubated for one hour at 37° C. Wells were washed sixtimes with PBS/Tween 20. Anti-ANGPTL3 antibody was detected by adding 50μl of TMB (tetramethyl benzidine) solution (ImmunoPure® TMB SubstrateKit, Pierce #34021) to the wells for 5 to 10 minutes. Plates were readspectrophotometrically at 450 nm using a microplate reader (MolecularDevices, Sunnyvale, Calif.).

H. Epitope Mapping of Monoclonal Antibodies

1. Antibody Binding to Certain ANGPTL3 Peptides

Six antibodies from cohort 1 were tested by antibody capture ELISA forbinding to three different 50 amino acid peptides from the N-terminus ofmouse ANGPTL3 and to N′-mANGPTL3T. The antibodies tested were designated1.251.1, 1.132.1, 1.173.2, 1.315.1, 1.424.1, and 1.431.1. Thoseantibodies were tested for binding to an S¹⁷-G⁶⁶ peptide (SEQ ID NO:11), a D⁴²-E⁹¹ peptide (SEQ ID NO: 12), a Q⁶⁷-M¹¹⁶ peptide (SEQ ID NO:13), and N′-mANGPTL3T (SEQ ID NO: 7).

The results of that experiment are shown in Table 2.

TABLE 2 Antibody binding to ANGPTL3 peptides mouse ANGPTL3 peptideantibody S¹⁷-G⁶⁶ D⁴²-E⁹¹ Q⁶⁷-M¹¹⁶ N′-mANGPTL3T 1.251.1 − + − + 1.132.1 −− − − 1.173.2 − + + + 1.315.1 − + − + 1.424.1 − + − + 1.431.1 − − − −

None of the tested antibodies bound to the S¹⁷-G⁶⁶ peptide. Antibodies1.251.1, 1.173.1, 1.315.1, and 1.424.1 bound to both the D⁴²-E⁹¹ peptideand N′-mANGPTL3T. Antibody 1.173.1 also bound to the Q⁶⁷-M¹¹⁶ peptide.Antibodies 1.132.1 and 1.431.1 did not bind to any of the peptidestested or to N′-mANGPTL3T, which suggests that those antibodies bind toan epitope outside of the region between S¹⁷ and D²⁴⁰ of mANGPTL3.

Eight antibodies from cohort 3 were tested by antibody capture ELISA forbinding to a synthetic peptide with the sequence of the SP1 region ofANGPTL3 (referred to as ANGPTL3 SP1) EPKSRFAMLDDVKILANGLLQLGHGL (SEQ IDNOS:9 and 10) and full-length HIS-tagged human ANGPTL3T:MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFEGHHHHHH (SEQ ID NO:4) andfull-length human ANGPTL3 (using ANGPTL3T, produced as described above)(SEQ ID NO:3). Unrelated recombinant protein was used as a control.

The results of that experiment are shown in FIG. 9. Antibodies 4.1.1,4.4.1, 4.8.1, and 4.8.3 bound to ANGPTL3 SP1 better than to full-lengthANGPTL3T. Antibodies 4.7.1 and 4.9.1 bound to both ANGPTL3 SP1 andANGPTL3T. Antibody 4.6.3 bound to full-length ANGPTL3T, but not toANGPTL3 SP1. Finally, antibody 4.8.2 bound to all proteins tested,including the control protein.

Antibodies from cohort 4 were tested for binding to hANGPTL3T andhANGPTL3 SP1 peptide (data not shown). Antibodies 5.35 and 5.50 bound toboth antigens and were selected for further analysis.

2. Alanine-Scanning Mutagenesis of SP1 Peptide

The epitopes for antibodies 4.7.1, 4.8.3, 4.9.1, 5.35, and 5.50 werefurther defined using alanine-scanning mutagenesis of the SP1 peptide.The wild-type SP1 peptide has the sequence EPKSRFAMLDDVKILANGLLQLGHGL(SEQ ID NO: 9). A series of 26 mutants was made, in which each of the 26amino acids of the wild-type SP1 peptide was changed to an alanine, asshown in Table 3:

TABLE 3 Mutant SP1 peptides peptide sequence SEQ ID NO: SP1EPKSRFAMLDDVKILANGLLQLGHGL 9 mutant 1 APKSRFAMLDDVKILANGLLQLGHGL 83mutant 2 EAKSRFAMLDDVKILANGLLQLGHGL 84 mutant 3EPASRFAMLDDVKILANGLLQLGHGL 85 mutant 4 EPKARFAMLDDVKILANGLLQLGHGL 86mutant 5 EPKSAFAMLDDVKILANGLLQLGHGL 87 mutant 6EPKSRAAMLDDVKILANGLLQLGHGL 88 “mutant 7” EPKSRFAMLDDVKILANGLLQLGHGL 9mutant 8 EPKSRFAALDDVKILANGLLQLGHGL 89 mutant 9EPKSRFAMADDVKILANGLLQLGHGL 90 mutant 10 EPKSRFAMLADVKILANGLLQLGHGL 91mutant 11 EPKSRFAMLDAVKILANGLLQLGHGL 92 mutant 12EPKSRFAMLDDAKILANGLLQLGHGL 93 mutant 13 EPKSRFAMLDDVAILANGLLQLGHGL 94mutant 14 EPKSRFAMLDDVKALANGLLQLGHGL 95 mutant 15EPKSRFAMLDDVKIAANGLLQLGHGL 96 “mutant 16” EPKSRFAMLDDVKILANGLLQLGHGL 9mutant 17 EPKSRFAMLDDVKILAAGLLQLGHGL 97 mutant 18EPKSRFAMLDDVKILANALLQLGHGL 98 mutant 19 EPKSRFAMLDDVKILANGALQLGHGL 99mutant 20 EPKSRFAMLDDVKILANGLAQLGHGL 100 mutant 21EPKSRFAMLDDVKILANGLLALGHGL 101 mutant 22 EPKSRFAMLDDVKILANGLLQAGHGL 102mutant 23 EPKSRFAMLDDVKILANGLLQLAHGL 103 mutant 24EPKSRFAMLDDVKILANGLLQLGAGL 104 mutant 25 EPKSRFAMLDDVKILANGLLQLGHAL 105mutant 26 EPKSRFAMLDDVKILANGLLQLGHGA 106“Mutant 7” and “Mutant 16” “replaced” an alanine in the wild-typesequence with an alanine. Those “mutants” therefore had the wild-typeSP1 peptide sequence and were not made.

Antibodies 4.7.1, 4.8.3, 4.9.1, 5.35, and 5.50 were tested by antibodycapture ELISA for binding to the synthetic peptides shown in Table 3(except for the “Mutant 7” and “Mutant 16” peptides), as follows.Alanine mutants were generated using a Flag-SUMO-SP1 gene cassette bypoint mutagenesis PCR. “Flag” is a flag tag and “SUMO” is a smallubiquitin-related modifier. Each mutant was in vitro translated and theconcentrations were normalized by western blotting with anti-flag M1monoclonal antibody. Antibody binding to each mutant was determined byan ELISA method as follows. The wild-type SP1 peptide and each mutantpeptide were separately captured in the wells of an ELISA plate that hadbeen coated with anti-flag M1 monoclonal antibody. A biotinylatedantibody (4.7.1, 4.8.3, 4.9.1, 5.35, or 5.50) was then added to thewells. The binding of the antibody to the peptide was detected withHRP-conjugated streptavidin (Pierce #21124), developed using TMB asdiscussed in Example G.

The results of that experiment are shown in FIG. 14. In each of the bargraphs in that Figure, the wild-type SP1 peptide is designated “0”, andis located at the far right of each bar graph. The epitope forantibodies 4.7.1 and 4.9.1 appeared to include amino acids 2 to 6 and 9of the SP1 peptide (mouse ANGPTL3 amino acids 33 to 37 and 40; humanANGPTL3 amino acids 33 to 37 and 40) in that experiment. The epitope forantibodies 5.35 and 5.50 appeared to include amino acids 3 to 6 and 9 to11 of the SP1 peptide (mouse ANGPTL3 amino acids 34 to 37 and 40 to 42;human ANGPTL3 amino acids 34 to 37 and 40 to 42) in that experiment. Theepitope for antibody 4.8.3 appeared to include amino acids 3 to 6, 9,and 11 of the SP1 peptide (mouse ANGPTL3 amino acids 34 to 37, 40, and42; human ANGPTL3 amino acids 34 to 37, 40, and 42) in that experiment.Thus, neutralizing antibodies 4.7.1, 4.8.3, 4.9.1, 5.35, and 5.50 allappeared to bind to an epitope within the SP1 region of ANGPTL3 thatincludes amino acids 34 to 37 and 40 of ANGPTL3.

I. Isotyping

The isotypes of antibodies 4.7.1, 4.8.3, 4.9.1, 5.35, 5.50, and 1.315.1were determined by standard methods. 4.7.1 and 5.50 are of the IgG2bisotype; 4.8.3, 1.315.1, and 5.35 are of the IgG2a isotype, and 4.9.1 isof the IgG1 isotype.

The predicted half-life in serum of an IgG2b antibody is 3 to 3.5 days.The predicted half-life in serum of an IgG2a antibody is 6 to 12 days.The predicted half-life in serum of an IgG1 antibody is 8 to 11 days.

J. In Vivo Administration of Monoclonal Antibodies to ANGPTL3

Monoclonal antibodies to ANGPTL3 were administered to mice to determineif such antibodies could effectively reduce triglyceride and/orcholesterol levels in mice. In the first experiment, 8 week old C57albino mice fed a standard diet (“chow-fed” mice) were injected with 30μg of a monoclonal antibody in a volume of 10 μl per gram of bodyweight. The anti-ANGPTL3 antibodies tested in that experiment were1.315.1, 4.7.1, 4.8.3, 4.9.1. An anti-KLH antibody was administered as acontrol. Five mice were injected for each antibody. Fed and fasted serumlevels of triglycerides and cholesterol were measured after four days.

The results of that experiment are shown in FIGS. 1 and 2. FIG. 1 showsfed and fasted triglyceride levels after four days in mice administered1.315.1, 4.7.1, 4.8.3, 4.9.1, and anti-KLH. When antibody 4.7.1 waspurified, two peaks were collected. Each of those peaks was administeredto the mice separately (4.7.1-P1 and 4.7.1-P2). All four anti-ANGPTL3antibodies caused a statistically significant decrease in serumtriglyceride levels in mice in the fed state when compared to miceadministered anti-KLH. In that experiment, however, none of theanti-ANGPTL3 antibodies caused a decrease in serum triglyceride levelsin mice in the fasted state. In fact, antibody 4.7.1 caused an increasein serum triglyceride levels in fasted mice in that experiment.

FIG. 2 shows fed and fasted cholesterol levels after four days in miceadministered 1.315.1, 4.7.1, 4.8.3, 4.9.1, and anti-KLH. As above, twoseparate peaks of antibody 4.7.1 were tested in that experiment. Again,all of the antibodies caused a statistically significant decrease inserum cholesterol levels in mice in the fed state when compared to miceadministered anti-KLH. In that experiment, however, none of theanti-ANGPTL3 antibodies caused a decrease in serum cholesterol levels inmice in the fasted state.

In the second experiment, chow-fed 8 week old C57 albino mice wereinjected with 30 μg of a monoclonal antibody in a volume of 10 μl pergram of body weight. Anti-ANGPTL3 antibody 4.8.3 was tested in thatexperiment. An anti-KLH antibody was administered as a control. Inaddition, anti-ANGPTL4 antibody 14D12 was tested for comparison. See,e.g., PCT Publication No. WO 2006/074228. Five mice were injected foreach antibody. Fed and fasted serum levels of triglycerides andcholesterol were measured after four days.

The results of that experiment are shown in FIGS. 3 and 4. FIG. 3 showsfed and fasted serum triglyceride levels in mice administeredanti-ANGPTL3 antibody 4.8.3, anti-ANGPTL4 antibody 14D12, or anti-KLHantibody. In that experiment, 4.8.3 reduced serum triglyceride levels bya statistically significant extent in both fed and fasted mice. Antibody14D12, on the other hand, reduced serum triglyceride levels by astatistically significant extent only in fasted mice in that experiment.

FIG. 4 shows fed and fasted serum cholesterol levels in miceadministered anti-ANGPTL3 antibody 4.8.3, anti-ANGPTL4 antibody 14D12,or anti-KLH antibody. In that experiment, 4.8.3 reduced serumcholesterol levels by a statistically significant extent in both fed andfasted mice. Antibody 14D12, on the other hand, reduced serumcholesterol levels by a statistically significant extent only in fastedmice in that experiment.

In the third experiment, 8 week old C57 albino mice fed a standard diet(“chow-fed” mice) were injected with 30 μg of a monoclonal antibody in avolume of 10 μl per gram of body weight. The anti-ANGPTL3 antibodiestested in that experiment were 4.7.1, 4.8.3, and 4.9.1. An anti-KLHantibody was administered as a control. In addition, anti-ANGPTL4antibody 14D12 was tested for comparison. Ten mice were injected foreach antibody. Fed and fasted serum levels of triglycerides andcholesterol were measured after four days.

The results of that experiment are shown in FIGS. 5 and 6. FIG. 5 showsfed and fasted triglyceride levels after four days in mice administered4.7.1, 4.8.3, 4.9.1, 14D12, and anti-KLH. Anti-ANGPTL3 antibodies 4.8.3and 4.9.1 caused a statistically significant decrease in serumtriglyceride levels in mice in the fed state in that experiment. Thefailure of antibody 4.7.1 to reduce triglycerides in the fed state maybe due, in part, to it being an IgG2b antibody, which has a predictedhalf-life in serum of only 3 to 3.5 days. Anti-ANGPTL3 antibodies 4.7.1,4.8.3, and 4.9.1, as well as anti-ANGPTL4 antibody 14D12 caused adecrease in serum triglyceride levels in mice in the fasted state inthat experiment.

FIG. 6 shows fed and fasted cholesterol levels after four days in miceadministered 4.7.1, 4.8.3, 4.9.1, 14D12, and anti-KLH. Anti-ANGPTL3antibodies 4.7.1, 4.8.3, and 4.9.1, as well as anti-ANGPTL4 antibody14D12 caused a decrease in serum cholesterol levels in mice in the fedstate in that experiment. In that experiment, however, only anti-ANGPTL3antibody 4.9.1 and anti-ANGPTL4 antibody 14 D12 caused a decrease inserum cholesterol levels in mice in the fasted state.

In the fourth experiment, 8 week old C57 albino mice fed a standard diet(“chow-fed” mice) were injected with 30 μg of a monoclonal antibody in avolume of 10 μl per gram of body weight. The anti-ANGPTL3 antibodiestested in that experiment were 1.125.1, 1.132.1, 1.173.2, 1.315.1,1.424.1, and 1.431.1. An anti-KLH antibody was administered as acontrol. There were five mice in each group. Fed and fasted serum levelsof triglycerides were measured after four days and after 8 days.

The results of that experiment are shown in FIG. 7. In that experiment,1.315.1 reduced serum triglycerides by a statistically significantextent after both 4 days and 8 days.

In a fifth experiment, 8 to 10 week old C57 albino mice fed a standarddiet (“chow-fed” mice) were injected with 0 μg, 3 μg, 10 μg, 30 μg, or90 μg of a monoclonal antibody in a volume of 10 μl per gram of bodyweight. The anti-ANGPTL3 antibodies tested in that experiment were 5.35and 5.50. Five mice were injected for each antibody at each dose. Fedserum levels of triglycerides and cholesterol were measured after fourdays and after seven days.

The results of that experiment are shown in FIGS. 15 and 16. FIG. 15shows serum triglycerides after four and seven days in mice injectedwith various amounts of antibodies 5.35 and 5.50. In that experiment,both antibodies 5.35 and 5.50 reduced serum triglycerides after fourdays in mice injected with 30 mg/kg or 90 mg/kg of antibody. Antibody5.50 also reduced serum triglycerides after four days in mice injectedwith 3 mg/kg or 10 mg/kg of antibody in that experiment. Serumtriglycerides were still reduced after seven days in mice injected with10 mg/kg, 30 mg/kg, or 90 mg/kg of antibody 5.50 in that experiment,while only mice injected with 90 mg/kg of antibody 5.35 continued tohave reduced serum triglycerides after seven days.

FIG. 16 shows serum cholesterol levels in mice injected with variousamounts of antibodies 5.35 and 5.50. In that experiment, mice injectedwith 30 mg/kg or 90 mg/kg of antibody 5.35, or 10 mg/kg, 30 mg/kg, or 90mg/kg of antibody 5.50 had reduced serum cholesterol levels after fourdays. Mice injected with 90 mg/kg of antibody 5.35 or 90 mg/kg ofantibody 5.50 continued to have reduced serum cholesterol levels afterseven days.

K. Administration of Monoclonal Antibodies Against ANGPTL3 in ApoEKnockout Mice

ApoE knockout mice have been found to develop spontaneoushypercholesterolemia. See, e.g., Piedrahita et al. (1992) Proc. Natl.Acad. Sci. USA 89(10):4471-5; and Zhang et al. (1992) Science258(5081):468-71. To determine if certain monoclonal antibodies againstANGPTL3 can reduce serum cholesterol and triglyceride levels in ApoEknockout mice, the following experiment was performed. Three groups ofeight 14-week old ApoE knockout mice (Taconic Animal Models, strainB6.129P2-Apoe^(tm1Unc)N11) were injected with 30 mg/kg anti-KLH, 30mg/kg antibody 5.35, or 30 mg/kg antibody 5.50 intraperitoneally. Eachmouse received one injection on day 0 and one injection on day 4. Allmice were fed a standard diet (“chow-fed”). Fed serum triglyceridelevels and fed cholesterol levels were determined in each mouse at day 0(pre-injection), day 4, day 8, and day 12.

The results of that experiment are shown in FIG. 19. Antibody 5.35reduced serum triglyceride levels by 36% by day 4. That reductionpersisted through day 8, but disappeared by day 12. Antibody 5.50reduced serum triglyceride levels by 60% by day 4. That reductionpersisted through day 8, and serum triglycerides remained significantlyreduced through day 12.

Antibody 5.35 reduced serum cholesterol levels by 17%, although thatreduction was not achieved until day 8 in that experiment, and thereduction diminished somewhat by day 12. Antibody 5.50 reduced serumcholesterol levels by day 4, and continued to reduce the serumcholesterol levels through day 8 and day 12, to a total reduction of 30%at the time that experiment was ended.

Those results showed that antibodies 5.35 and 5.50 reduced both serumtriglyceride levels and serum cholesterol levels in ApoE mice. Antibody5.50 was more effective at reducing both serum triglyceride levels andserum cholesterol levels in that experiment, which may be due to thelonger half-life of that antibody.

L. In Vitro Administration of Monoclonal Antibodies to ANGPTL3

The effect of mouse monoclonal anti-ANGPTL3 on LPL activity was testedusing an in vitro assay. To obtain LPL-conditioned media, HEK293F cells(the FreeStyle™ 293 Expression System) (Invitrogen, Carlsbad, Calif.)were transfected with an LPL expression vector comprising DNA encodingLPL with a C-terminal FLAG tag in the IRES puro plasmid (Clontech,Mountain View, Calif.).

Transformed cells were grown in selection medium (FreeStyle™ 293 Systemmedium (GIBCO BRL, Gaithersburg, Md.) supplemented with 1×Penicillin-Streptomycin-Glutamine (Invitrogen, Carlsbad, Calif.) and 2.4μg/ml of puromycin). Cells were enumerated and pelleted bycentrifugation. The resulting cell pellet was resuspended in freshFreeStyle™ 293 medium containing 1×GPS, without puromycin) at aconcentration of 1×10⁶ cells/ml. Following a 48 hour incubation,conditioned medium containing LPL was harvested from the cells,distributed in 5 ml aliquots and then frozen at −70° C. until used.

LPL activity in conditioned medium was assayed by adding 200 mg/dLIntralipid® (Pharmacia & Upjohn), 12 mM Glucose, 5 units/ml heparin and200 μl 5% mouse serum (as a source of Apo C-II) to aliquots ofconditioned medium. That medium was then incubated at 37° C. Sampleswere harvested at 2 hours, 4 hours, and 6 hours and frozen immediatelyat −70° C. until assays were performed. LPL conditioned medium wasassayed for LPL activity by measuring FFA levels using undilutedconditioned medium and dilutions of 1:2, 1:4, 1:8, 1:16, and 1:32. TheFFA levels were determined at 2 hours, 4 hours and 6 hour using a WakoFFA kit (Wako Chemical USA, Inc., Richmond, Va.).

The ability of monoclonal antibodies to neutralize ANGPTL3 activity inan in vitro assay for LPL activity was also determined. The in vitroassay for LPL activity measures FFA levels using a Wako FFA kit (WakoChemical USA, Inc., Richmond, Va.). Separate LPL activity assays wereconducted in the presence of 25 nM mouse ANGPTL3 and four concentrations(0.8 μg/ml, 2 μg/ml, 10 μg/ml and 50 μg/ml) of each of 5 monoclonalantibodies (antibodies 4.9.1, 4.8.3, 1.315.1, 4.7.1, and 1.173.2). Theresults are shown in FIG. 12. For each antibody, neutralizing activityis demonstrated by the antibody's ability to increase LPL activity,i.e., to “rescue” LPL from inhibition by ANGPTL3. Rescuing activity wasdetermined as the percentage increase in LPL activity in the presence ofboth ANGPTL3 and anti-ANGPTL3 antibody relative to LPL activity in thepresence of ANGPTL3 alone. Four antibodies (4.9.1, 4.8.3, 1.315.1 and4.7.1) were able to rescue LPL activity by over 50% depending on theirconcentration. This result indicated that those antibodies were able torescue LPL activity by neutralizing ANGPTL3 activity. In contrast,antibody 1.173.2 did not demonstrate significant rescue of LPL activityand as such served as an internal negative control.

M. Binding Affinity of Monoclonal Antibodies Against ANGPTL3

The affinity constants of antibodies 4.7.1, 4.8.3, and 4.9.1 forN′-hANGPTL3T and hANGPTL3T and the affinity constants of antibodies4.7.1, 4.9.1, 5.35, and 5.50 to the SP1 peptide were determined using aBIACORE® 3000 system (GE Healthcare, Uppsala, Sweden). The BIACORE® 3000is a real-time biomolecular analysis system for detecting andquantifying protein-protein interactions using surface plasmon resonancetechnology. The following affinity constants were determined accordingto the manufacturer's instructions for the BIACORE® 3000 system:equilibrium dissociation constant (K_(D)), association rate constant(k_(on)), and dissociation rate constant (k_(off)).

Affinity constants of the 4.7.1, 4.8.3, and 4.9.1 antibodies forN′-hANGPTL3T and hANGPTL3T protein antigens were each determined asfollows. The BIACORE CM5 chip surface was coated with a protein antigenby coupling either 10 μg/ml of N′-hANGPTL3T or 20 μg/ml of hANGPTL3T tothe chip surface using an Amine Coupling Kit (Product No.: BR-1000-50,GE Healthcare, Uppsala Sweden). The association and dissociation phases,which are used to determine the k_(on) and k_(off) constants, weregenerated by first injecting a Fab fragment of the antibody (generatedusing a Fab Preparation Kit (Product No.: 44885, Pierce, Rockford, Ill.61105) according to the manufacturer's instructions) into the BIACORE®3000 system having a coated chip surface for 2 minutes at a flow rate of30 μl/min. The concentrations of Fab fragment tested were 200 nM, 100nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5 625 nM, 0.78125 nM,and 0 nM. The injection phase was used to determine the association rateconstant, or k_(on). Following each injection, the Fab fragment wasdissociated from the protein antigen by flowing BIACORE HBS-EP buffer(Product No.: BR-1001-88, GE Healthcare, Uppsala Sweden) for 5 minutesover the antigen-Fab complex on the BIACORE CM5 chip surface. Thisdissociation phase was used to determine the dissociation rate constant,or k_(off). Prior to the next injection, the BIACORE chip surface wasregenerated by injecting 10 mM HCl at a flow rate of 100 μl/min for 30seconds. BIACORE sensograms for data analysis were generated bysubtracting the non-specific binding profile of a reference surface(immobilized with equivalent levels of unrelated proteins) from theFab-specific binding profiles of 4.7.1, 4.8.3, and 4.9.1 surfaces. Theaffinity parameters, including the K_(D), k_(on), and k_(off) values,were determined by globally fitting the association and dissociationdata sets to 1:1 binding model with mass transfer correction usingBIAevalution Software Version 3.0 (GE Healthcare, Uppsala Sweden).

Affinity constants of the 4.7.1, 4.9.1, 5.35, and 5.50 antibodies forthe SP1 peptide antigen were each determined as follows. The BIACORE CM5chip surface was coated with an anti-mouse IgG F_(c) by coupling 90μg/ml of Immunopure Goat Anti-Mouse IgG F_(c) (Product No.: 31170,Pierce, Rockford, Ill. 61105) to the chip surface using an AmineCoupling Kit (Product No.: BR-1000-50, GE Healthcare, Uppsala Sweden).The antibody was then captured on the anti-mouse IgG F_(c) BIACORE chipsurface by injecting the antibody at a flow rate of 10 μl/min for 30sec. The association and dissociation phases, which are used todetermine k_(on) and k_(off) constants, were generated by firstinjecting the SP1 peptide into the BIACORE® 3000 system having anantibody-coated chip surface for 2 minutes at a flow rate of 30 μl/min.The concentrations of SP1 peptide tested were 200 nM, 100 nM, 50 nM, 25nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, and 0 nM. Theinjection phase was used to determine the association rate constant, ork_(on). Following each injection, the SP1 peptide was dissociated fromthe antibody by flowing BIACORE HBS-EP buffer (Product No.: BR-1001-88,GE Healthcare, Uppsala Sweden) for 5 minutes over the peptide-antibodycomplex on the BIACORE CM5 chip surface. This dissociation phase wasused to determine the dissociation rate constant, or k_(off). Prior tothe next injection, the BIACORE chip surface was regenerated byinjecting 10 mM HCl at a flow rate of 100 μl/min for 30 seconds. BIACOREsensograms for data analysis were generated by subtracting thenon-specific binding profile of a reference surface (capturingequivalent levels of unrelated mouse IgG) from the antigen-specificbinding profiles of the 4.7.1, 4.9.1, 5.35, and 5.50 surfaces. Theaffinity parameters, including the K_(D), k_(on), and k_(off) values,were determined by globally fitting the association and dissociationdata sets to 1:1 binding model with mass transfer correction usingBIAevalution Software Version 3.0 (GE Healthcare, Uppsala Sweden).

The results of that experiment are shown in Tables 4, 5, and 6. Eachantibody was tested in duplicate, and the results for each experimentare shown.

TABLE 4 Antibody affinities for N′-hANGPTL3T. Antibody K_(D) (nM) k_(on)(M⁻¹sec⁻¹) k_(off) (sec⁻¹) 4.7.1 49.5 1.85 × 10⁵ 9.15 × 10⁻³ 4.7.1 77.51.16 × 10⁵ 8.99 × 10⁻³ 4.8.3 210 9.00 × 10⁴ 1.90 × 10⁻² 4.8.3 251 1.09 ×10⁵ 2.75 × 10⁻² 4.9.1 45.4 1.31 × 10⁵ 5.93 × 10⁻³ 4.9.1 55 1.33 × 10⁵7.34 × 10⁻³

TABLE 5 Antibody affinities for hANGPTL3T. Antibody K_(D) (nM) k_(on)(M⁻¹sec⁻¹) k_(off) (sec⁻¹) 4.7.1 45.3 1.76 × 10⁵ 7.96 × 10⁻³ 4.7.1 1178.12 × 10⁴ 9.53 × 10⁻³ 4.8.3 970 2.23 × 10⁴ 2.16 × 10⁻² 4.8.3 450 6.00 ×10⁴ 2.73 × 10⁻² 4.9.1 48.1 1.29 × 10⁵ 6.22 × 10⁻³ 4.9.1 45.2 1.72 × 10⁵7.77 × 10⁻³

TABLE 6 Antibody affinities for SP1 peptide. Antibody K_(D) (nM) k_(on)(M⁻¹sec⁻¹) k_(off) (sec⁻¹) 4.7.1 26.1 2.09 × 10⁵ 5.46 × 10⁻³ 4.9.1 46.11.69 × 10⁵ 7.78 × 10⁻³ 5.35 1.80 8.35 × 10⁵  1.5 × 10⁻³ 5.50 1.78 1.08 ×10⁶ 1.93 × 10⁻³

N. In Vivo Pharmacokinetics of Certain Monoclonal Antibodies AgainstANGPTL3

To determine the pharmacokinetics of these antibodies (antibodies 4.7.1,4.8.3, and 4.9.1) in vivo, each was administered separately to fourdifferent mice by intraperitoneal injection, at a dose of 30 mg per kg.At the time points indicated in FIG. 13, the mice were bled and serumwas obtained. The levels of anti-ANGPTL3 antibody present in the serumwas determined by comparing titers determined by antibody capture ELISAto a standard curve established using serial dilutions of the sameantibody that was injected into the mouse in an antibody capture ELISA.The antibody capture ELISAs were performed as described in Example G.The results can be seen in FIG. 13.

To determine the pharmacokinetics of antibodies 5.35 and 5.50 in vivo,antibody levels were determined in the mice described in Example J,fifth experiment, at 4 days, 7 days, and 12 days after injection. Thelevels of anti-ANGPTL3 antibody present in the serum was determined byantibody capture ELISA using recombinant human ANGPTL3-coated plates.The concentration of antibody in the serum was determined by comparingto a standard curve established using serial dilutions of the sameantibody that was injected into the mouse in an antibody capture ELISA.The ELISAs were performed as described in Example G.

The results of that experiment are shown in FIG. 17. In that experiment,antibody 5.50 had a longer half-life in vivo than antibody 5.35. Thelonger half-life correlated with the increased efficacy in vivo ofantibody 5.50 compared to antibody 5.35 in the experiment discussed inExample J, and as shown in FIGS. 15 and 16.

The pharmacokinetics of antibodies 4.7.1, 4.9.1, 5.35, and 5.50 in C57mice after a single injection of 30 mg/kg antibody is shown in FIG. 18.The antibody concentration was determined by antibody capture ELISA,performed as described in Example G. The concentration of antibody inthe serum was determined by comparing to a standard curve establishedusing serial dilutions of the same antibody that had been injected intothe mouse in an antibody capture ELISA. In that experiment, antibodies5.50 and 4.9.1 had approximately equivalent half-lives. In addition, thehalf-lives of antibodies 5.50 and 4.9.1 were longer than the half-livesof antibodies 5.35 and 4.7.1, which were approximately equivalent toeach other in that experiment. Furthermore, the Cmax of antibodies 5.35and 5.50 were approximately equivalent, and were greater than the Cmaxof antibody 4.9.1, which in turn was greater than the Cmax of antibody4.7.1, in that experiment.

O. Sequences of Certain Monoclonal Antibodies Against ANGPTL3

The heavy chain and light chain variable regions of antibodies 4.7.1,4.8.3, 4.9.1, 5.35, and 5.50 were determined. The heavy chain variableregion of antibody 4.7.1 is shown in SEQ ID NO: 19 (with the N-terminalsignal peptide) and in SEQ ID NO: 20 (without the N-terminal signalpeptide). The light chain variable region of antibody 4.7.1 is shown inSEQ ID NO: 27 (with the N-terminal signal peptide) and in SEQ ID NO: 28(without the N-terminal signal peptide). The heavy chain variable regionof antibody 4.8.3 is shown in SEQ ID NO: 21 (with the N-terminal signalpeptide) and in SEQ ID NO: 22 (without the N-terminal signal peptide).The light chain variable region of antibody 4.8.3 is shown in SEQ ID NO:29 (with the N-terminal signal peptide) and in SEQ ID NO: 30 (withoutthe N-terminal signal peptide). The heavy chain variable region ofantibody 4.9.1 is shown in SEQ ID NO: 23 (with the N-terminal signalpeptide) and in SEQ ID NO: 24 (without the N-terminal signal peptide).The light chain variable region of antibody 4.9.1 is shown in SEQ ID NO:31 (with the N-terminal signal peptide) and in SEQ ID NO: 32 (withoutthe N-terminal signal peptide). The heavy chain variable region ofantibody 5.35 is shown in SEQ ID NO: 63 (with the N-terminal signalpeptide) and in SEQ ID NO: 64 (without the N-terminal signal peptide).The light chain variable region of antibody 5.35 is shown in SEQ ID NO:67 (with the N-terminal signal peptide) and in SEQ ID NO: 68 (withoutthe N-terminal signal peptide). The heavy chain variable region ofantibody 5.50 is shown in SEQ ID NO: 65 (with the N-terminal signalpeptide) and in SEQ ID NO: 66 (without the N-terminal signal peptide).The light chain variable region of antibody 5.50 is shown in SEQ ID NO:69 (with the N-terminal signal peptide) and in SEQ ID NO: 70 (withoutthe N-terminal signal peptide).

An alignment of the heavy chain variable regions of antibodies 4.7.1,4.8.3, and 4.9.1 is shown in FIG. 10. The consensus sequence for theheavy chain variable regions is also shown (SEQ ID NO: 25). Theconsensus sequence for the heavy chain variable regions without thesignal peptide is shown in Table 7 (SEQ ID NO: 26).

An alignment of the light chain variable regions of antibodies 4.7.1,4.8.3, and 4.9.1 is shown in FIG. 11. The consensus sequence for thelight chain variable regions is also shown (SEQ ID NO: 33). Theconsensus sequence for the light chain variable regions without thesignal peptide is shown in Table 7 (SEQ ID NO: 34).

P. Humanization of Certain Monoclonal Antibodies Against ANGPTL3

The following protocol describes humanization of antibody 4.7.1.Antibodies 4.8.3, 4.9.1, 5.35, 5.50, and 1.315.1 can be humanized by thesame method. The humanized version of antibody 4.7.1 is referred to as“hu4.7.1.” The humanized versions of antibodies 4.8.3, 4.9.1, 5.35,5.50, and 1.315.1, are referred to as “hu4.8.3,” “hu4.9.1,” “hu5.35,”“hu5.50,” and “hu1.315.1,” respectively.

Human framework regions for each of the heavy chain and light chain areselected from a set of family-specific consensus human framework regionsbased on their homology to the mouse framework regions present inantibody 4.7.1. The selected human framework regions are diversified atspecific amino acid positions to reflect framework diversity within thechosen family of V-genes. Polynucleotides encoding CDR1, CDR2, and CDR3of the heavy chain of antibody 4.7.1 are cloned into the library ofpolynucleotides that encode the diversified human framework regions forthe heavy chain. The resulting library is referred to as a humanized4.7.1 heavy chain variable region library. Polynucleotides encodingCDR1, CDR2, and CDR3 of the light chain of antibody 4.7.1 are clonedinto the library of polynucleotides that encode the diversified humanframework regions for the light chain. The resulting library is referredto as a humanized 4.7.1 light chain variable region library. Thehumanized 4.7.1 heavy chain variable region library and the humanized4.7.1 light chain variable region library are cloned into a phagedisplay vector in a single chain Fv (scFv) format.

The scFv phage display library is then screened against the targetantigen, e.g., human ANGPTL3, through 2-3 rounds of binding to selectfor high affinity scFvs. The scFvs are then expressed in soluble formand tested for target affinity and/or in vitro neutralizing potency. Theheavy chain variable region and light chain variable region of the scFvsselected for suitable affinity and potency are then expressed asfull-length IgGs or as scFv-CL-PEG (CL is the human constant lightchain) and tested for in vivo activity, e.g., reduction in serumtriglycerides and/or serum cholesterol in mice. PEG is attached througha cysteine on the CL group.

Q. Affinity Maturation of Certain Monoclonal Antibodies Against ANGPTL3

The following protocol describes affinity maturation of antibody 4.7.1.Affinity maturation of antibodies 4.8.3, 4.9.1, 5.35, 5.50, and 1.315.1can be carried out using the same method. Similarly, affinity maturationof hu4.7.1, hu4.8.3, hu4.9.1, hu5.35, hu5.50, and hu1.315.1 can becarried out using the same method.

A polynucleotide encoding the heavy chain of antibody 4.7.1 is subjectedto random mutagenesis, e.g., using error-prone PCR. Error-prone PCR wasperformed using the GeneMorph® II Random Mutagenesis Kit (Stratagene, LaJolla, Calif.) according to the manufacturer's instructions.

A polynucleotide encoding the light chain of antibody 4.7.1 is alsosubjected to random mutagenesis, e.g., using error-prone PCR.Error-prone PCR was performed using the GeneMorph® II Random MutagenesisKit (Stratagene, La Jolla, Calif.) according to the manufacturer'sinstructions. The randomly mutated heavy chain polynucleotide and therandomly mutated light chain polynucleotide are cloned into a phagedisplay vector in a single chain Fv (scFv) format.

The scFv phage display library is then screened against the targetantigen, e.g., human ANGPTL3, through 2-3 rounds of binding to selectfor high affinity scFvs. The scFvs are then expressed in soluble formand tested for target affinity and/or in vitro neutralizing potency. Theheavy chain variable region and light chain variable region of the scFvsselected for suitable affinity and potency are then expressed asfull-length IgGs or as scFv-CL-PEG, and tested for in vivo activity,e.g., reduction in serum triglycerides and/or serum cholesterol in mice.

The selected affinity matured antibodies can then be humanized asdescribed above in Example P, if not already humanized.

While the above examples describe, inter alia, certain neutralizingmonoclonal antibodies against mouse ANGPTL3 and the in vivo effects ofthose antibodies in mice, one skilled in the art would readily recognizethat neutralizing monoclonal antibodies against human ANGPTL3 may begenerated, and such antibodies would have the same or similar in vivoeffects in humans. That conclusion is based, in part, on the observationthat human and mouse ANGPTL3 are evolutionarily conserved proteins thatshare structural and functional features. Conklin, D., et al. (1999)Genomics 62:477-482. For example, human and mouse ANGPTL3 share about76% amino acid sequence identity. Human and mouse ANGPTL3 also sharecommon secondary structural elements, e.g., an N-terminal coiled-coildomain and a C-terminal fibrinogen-like domain. Furthermore, humanANGPTL3 has a similar function as mouse ANGPTL3, as demonstrated by theability of human ANGPTL3 to raise serum lipid levels when overexpressedin mice. Koishi et al. (2002) Nat. Genet. 30(2):151-157.

It is generally recognized in the art that mice are routinely used asmodels for the treatment of various conditions and diseases usingneutralizing antibodies. For example, neutralizing antibodies have beenused to treat prion disease, diabetes, and inflammation in mice. See,e.g., White et al. (2003) Nature 422:80-83; Cailleau et al. (1997)Diabetes 46:937-940; and Lochner et al. (2002) J. Immunol. Methods259:149-157. In the latter study, monoclonal antibodies that neutralizemouse IL-18 were raised in IL-18 deficient mice. Those mouse monoclonalantibodies were capable of suppressing lipopolysaccharide-inducedinflammatory response in wild-type mice. Thus, one skilled in the artwould conclude that the foregoing examples support the use ofneutralizing monoclonal antibodies against human ANGPTL3 in thetreatment of human medical conditions.

TABLE 7 Table of Sequences SEQ ID Description NO: Sequence mouse ANGPTL31 MHTIKLFLFV VPLVIASRVD PDLSSFDSAP SEPKSRFAML (Accession No. DDVKILANGLLQLGHGLKDF VHKTKGQIND IFQKLNIFDQ NP_038941) SFYDLSLRTN EIKEEEKELRRTTSTLQVKN EEVKNMSVEL NSKLESLLEE KTALQHKVRA LEEQLTNLIL SPAGAQEHPEVTSLKSFVEQ QDNSIRELLQ SVEEQYKQLS QQHMQIKEIE KQLRKTGIQE PSENSLSSKSRAPRTTPPLQ LNETENTEQD DLPADCSAVY NRGEHTSGVY TIKPRNSQGF NVYCDTQSGSPWTLIQHRKD GSQDFNETWE NYEKGFGRLD GEFWLGLEKI YAIVQQSNYI LRLELQDWKDSKHYVEYSFH LGSHETNYTL HVAEIAGNIP GALPEHTDLM FSTWNHRAKG QLYCPESYSGGWWWNDICGE NNLNGKYNKP RTKSRPERRR GIYWRPQSRK LYAIKSSKMM LQPTT mANGPTL3T 2MHTIKLFLFV VPLVIASRVD PDLSSFDSAP SEPKSRFAML DDVKILANGL LQLGHGLKDFVHKTKGQIND IFQKLNIFDQ SFYDLSLRTN EIKEEEKELR RTTSTLQVKN EEVKNMSVELNSKLESLLEE KTALQHKVRA LEEQLTNLIL SPAGAQEHPE VTSLKSFVEQ QDNSIRELLQSVEEQYKQLS QQHMQIKEIE KQLRKTGIQE PSENSLSSKS RAPRTTPPLQ LNETENTEQDDLPADCSAVY NRGEHTSGVY TIKPRNSQGF NVYCDTQSGS PWTLIQHRKD GSQDFNETWENYEKGFGRLD GEFWLGLEKI YAIVQQSNYI LRLELQDWKD SKHYVEYSFH LGSHETNYTLHVAEIAGNIP GALPEHTDLM FSTWNHRAKG QLYCPESYSG GWWWNDICGE NNLNGKYNKPRTKSRPERRR GIYWRPQSRK LYAIKSSKMM LQPTTGHHHHH H human ANGPTL3 3MFTIKLLLFI VPLVISSRID QDNSSFDSLS PEPKSRFAML (Accession No. DDVKILANGLLQLGHGLKDF VHKTKGQIND IFQKLNIFDQ NP_055310) SFYDLSLQTS EIKEEEKELRRTTYKLQVKN EEVKNMSLEL NSKLESLLEE KILLQQKVKY LEEQLTNLIQ NQPETPEHPEVTSLKTFVEK QDNSIKDLLQ TVEDQYKQLN QQHSQIKEIE NQLRRTSIQE PTEISLSSKPRAPRTTPFLQ LNEIRNVKHD GIPAECTTIY NRGEHTSGMY AIRPSNSQVF HVYCDVISGSPWTLIQHRID GSQNFNETWE NYKYGFGRLD GEFWLGLEKI YSIVKQSNYV LRIELEDWKDNKHYIEYSFY LGNHETNYTL HLVAITGNVP NAIPENKDLV FSTWDHKAKG HFNCPEGYSGGWWWHDECGE NNLNGKYNKP RAKSKPERRR GLSWKSQNGR LYSIKSTKML IHPTDSESFEhANGPTL3T 4 MFTIKLLLFI VPLVISSRID QDNSSFDSLS PEPKSRFAML DDVKILANGLLQLGHGLKDF VHKTKGQIND IFQKLNIFDQ SFYDLSLQTS EIKEEEKELR RTTYKLQVKNEEVKNMSLEL NSKLESLLEE KILLQQKVKY LEEQLTNLIQ NQPETPEHPE VTSLKTFVEKQDNSIKDLLQ TVEDQYKQLN QQHSQIKEIE NQLRRTSIQE PTEISLSSKP RAPRTTPFLQLNEIRNVKHD GIPAECTTIY NRGEHTSGMY AIRPSNSQVF HVYCDVISGS PWTLIQHRIDGSQNFNETWE NYKYGFGRLD GEFWLGLEKI YSIVKQSNYV LRIELEDWKD NKHYIEYSFYLGNHETNYTL HLVAITGNVP NAIPENKDLV FSTWDHKAKG HFNCPEGYSG GWWWHDECGENNLNGKYNKP RAKSKPERRR GLSWKSQNGR LYSIKSTKML IHPTDSESFE GHHHHHH mouseAngptl3 5 TCAGGAGGGA GAAGTTCCAA ATTGCTTAAA ATTGAATAAT (Accession No.TGAGACAAAA AATGCACACA ATTAAATTAT TCCTTTTTGT NM_013913) TGTTCCTTTAGTAATTGCAT CCAGAGTGGA TCCAGACCTT (mRNA/cDNA) TCATCATTTG ATTCTGCACCTTCAGAGCCA AAATCAAGAT TTGCTATGTT GGATGATGTC AAAATTTTAG CGAATGGCCTCCTGCAGCTG GGTCATGGAC TTAAAGATTT TGTCCATAAG ACTAAGGGAC AAATTAACGACATATTTCAG AAGCTCAACA TATTTGATCA GTCTTTTTAT GACCTATCAC TTCGAACCAATGAAATCAAA GAAGAGGAAA AGGAGCTAAG AAGAACTACA TCTACACTAC AAGTTAAAAACGAGGAGGTG AAGAACATGT CAGTAGAACT GAACTCAAAG CTTGAGAGTC TGCTGGAAGAGAAGACAGCC CTTCAACACA AGGTCAGGGC TTTGGAGGAG CAGCTAACCA ACTTAATTCTAAGCCCAGCT GGGGCTCAGG AGCACCCAGA AGTAACATCA CTCAAAAGTT TTGTAGAACAGCAAGACAAC AGCATAAGAG AACTCCTCCA GAGTGTGGAA GAACAGTATA AACAATTAAGTCAACAGCAC ATGCAGATAA AAGAAATAGA AAAGCAGCTC AGAAAGACTG GTATTCAAGAACCCTCAGAA AATTCTCTTT CTTCTAAATC AAGAGCACCA AGAACTACTC CCCCTCTTCAACTGAACGAA ACAGAAAATA CAGAACAAGA TGACCTTCCT GCCGACTGCT CTGCCGTTTATAACAGAGGC GAACATACAA GTGGCGTGTA CACTATTAAA CCAAGAAACT CCCAAGGGTTTAATGTCTAC TGTGATACCC AATCAGGCAG TCCATGGACA TTAATTCAAC ACCGGAAAGATGGCTCACAG GACTTCAACG AAACATGGGA AAACTACGAA AAGGGCTTTG GGAGGCTCGATGGAGAATTT TGGTTGGGCC TAGAGAAGAT CTATGCTATA GTCCAACAGT CTAACTACATTTTACGACTC GAGCTACAAG ACTGGAAAGA CAGCAAGCAC TACGTTGAAT ACTCCTTTCACCTGGGCAGT CACGAAACCA ACTACACGCT ACATGTGGCT GAGATTGCTG GCAATATCCCTGGGGCCCTC CCAGAGCACA CAGACCTGAT GTTTTCTACA TGGAATCACA GAGCAAAGGGACAGCTCTAC TGTCCAGAAA GTTACTCAGG TGGCTGGTGG TGGAATGACA TATGTGGAGAAAACAACCTA AATGGAAAAT ACAACAAACC CAGAACCAAA TCCAGACCAG AGAGAAGAAGAGGGATCTAC TGGAGACCTC AGAGCAGAAA GCTCTATGCT ATCAAATCAT CCAAAATGATGCTCCAGCCC ACCACCTAAG AAGCTTCAAC TGAACTGAGA CAAAATAAAA GATCAATAAATTAAATATTA AAGTCCTCCC GATCACTGTA GTAATCTGGT ATTAAAATTT TAATGGAAAGCTTGAGAATT GAATTTCAAT TAGGTTTAAA CTCATTGTTA AGATCAGATA TCACCGAATCAACGTAAACA AAATTTATCT TTTTC human Angptl3 6 TTCCAGAAGA AAACAGTTCCACGTTGCTTG AAATTGAAAA (Accession No. TCAAGATAAA AATGTTCACA ATTAAGCTCCTTCTTTTTAT NM_014495) TGTTCCTCTA GTTATTTCCT CCAGAATTGA TCAAGACAAT(mRNA/cDNA) TCATCATTTG ATTCTCTATC TCCAGAGCCA AAATCAAGAT TTGCTATGTTAGACGATGTA AAAATTTTAG CCAATGGCCT CCTTCAGTTG GGACATGGTC TTAAAGACTTTGTCCATAAG ACGAAGGGCC AAATTAATGA CATATTTCAA AAACTCAACA TATTTGATCAGTCTTTTTAT GATCTATCGC TGCAAACCAG TGAAATCAAA GAAGAAGAAA AGGAACTGAGAAGAACTACA TATAAACTAC AAGTCAAAAA TGAAGAGGTA AAGAATATGT CACTTGAACTCAACTCAAAA CTTGAAAGCC TCCTAGAAGA AAAAATTCTA CTTCAACAAA AAGTGAAATATTTAGAAGAG CAACTAACTA ACTTAATTCA AAATCAACCT GAAACTCCAG AACACCCAGAAGTAACTTCA CTTAAAACTT TTGTAGAAAA ACAAGATAAT AGCATCAAAG ACCTTCTCCAGACCGTGGAA GACCAATATA AACAATTAAA CCAACAGCAT AGTCAAATAA AAGAAATAGAAAATCAGCTC AGAAGGACTA GTATTCAAGA ACCCACAGAA ATTTCTCTAT CTTCCAAGCCAAGAGCACCA AGAACTACTC CCTTTCTTCA GTTGAATGAA ATAAGAAATG TAAAACATGATGGCATTCCT GCTGAATGTA CCACCATTTA TAACAGAGGT GAACATACAA GTGGCATGTATGCCATCAGA CCCAGCAACT CTCAAGTTTT TCATGTCTAC TGTGATGTTA TATCAGGTAGTCCATGGACA TTAATTCAAC ATCGAATAGA TGGATCACAA AACTTCAATG AAACGTGGGAGAACTACAAA TATGGTTTTG GGAGGCTTGA TGGAGAATTT TGGTTGGGCC TAGAGAAGATATACTCCATA GTGAAGCAAT CTAATTATGT TTTACGAATT GAGTTGGAAG ACTGGAAAGACAACAAACAT TATATTGAAT ATTCTTTTTA CTTGGGAAAT CACGAAACCA ACTATACGCTACATCTAGTT GCGATTACTG GCAATGTCCC CAATGCAATC CCGGAAAACA AAGATTTGGTGTTTTCTACT TGGGATCACA AAGCAAAAGG ACACTTCAAC TGTCCAGAGG GTTATTCAGGAGGCTGGTGG TGGCATGATG AGTGTGGAGA AAACAACCTA AATGGTAAAT ATAACAAACCAAGAGCAAAA TCTAAGCCAG AGAGGAGAAG AGGATTATCT TGGAAGTCTC AAAATGGAAGGTTATACTCT ATAAAATCAA CCAAAATGTT GATCCATCCA ACAGATTCAG AAAGCTTTGAATGAACTGAG GCAAATTTAA AAGGCAATAA TTTAAACATT AACCTCATTC CAAGTTAATGTGGTCTAATA ATCTGGTATT AAATCCTTAA GAGAAAGCTT GAGAAATAGA TTTTTTTTATCTTAAAGTCA CTGTCTATTT AAGATTAAAC ATACAATCAC ATAACCTTAA AGAATACCGTTTACATTTCT CAATCAAAAT TCTTATAATA CTATTTGTTT TAAATTTTGT GATGTGGGAATCAATTTTAG ATGGTCACAA TCTAGATTAT AATCAATAGG TGAACTTATT AAATAACTTTTCTAAATAAA AAATTTAGAG ACTTTTATTT TAAAAGGCAT CATATGAGCT AATATCACAACTTTCCCAGT TTAAAAAACT AGTACTCTTG TTAAAACTCT AAACTTGACT AAATACAGAGGACTGGTAAT TGTACAGTTC TTAAATGTTG TAGTATTAAT TTCAAAACTA AAAATCGTCAGCACAGAGTA TGTGTAAAAA TCTGTAATAC AAATTTTTAA ACTGATGCTT CATTTTGCTACAAAATAATT TGGAGTAAAT GTTTGATATG ATTTATTTAT GAAACCTAAT GAAGCAGAATTAAATACTGT ATTAAAATAA GTTCGCTGTC TTTAAACAAA TGGAGATGAC TACTAAGTCACATTGACTTT AACATGAGGT ATCACTATAC CTTATT rN′-mANGPTL3T 7SRVDPD LSSFDSAPSE PKSRFAMLDD VKILANGLLQLGHGLKDFVH KTKGQINDIF QKLNIFDQSF YDLSLRTNEIKEEEKELRRT TSTLQVKNEE VKNMSVELNS KLESLLEEKTALQHKVRALE EQLTNLILSP AGAQEHPEVT SLKSFVEQQDNSIRELLQSV EEQYKQLSQQ HMQIKEIEKQ LRKTGIQEPSENSLSSKSRA PRTTPPLQLN ETENTEQDLE HHHHHH rN′-hANGPTL3T 8DQDNSS FDSLSPEPKS RFAMLDDVKI LANGLLQLGHGLKDFVHKTK GQINDIFQKL NIFDQSFYDL SLQTSEIKEEEKELRRTTYK LQVKNEEVKN MSLELNSKLE SLLEEKILLQQKVKYLEEQL TNLIQNQPET PEHPEVTSLK TFVEKQDNSIKDLLQTVEDQ YKQLNQQHSQ IKEIENQLRR TSIQEPTEISLSSKPRAPRT TPFLQLNEIR NVKHDGIPLE HHHHHH mANGPTL3 SP1 9EPKSRFAMLDDVKILANGLLQLGHGL region hANGPTL3 SP1 10EPKSRFAMLDDVKILANGLLQLGHGL region S¹⁷-G⁶⁶ 11SRVDPDLSSFDSAPSEPKSRFAMLDDVKILANGL peptide LQLGHGLKDFVHKTKG D⁴²-E⁹¹ 12DVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQ peptide SFYDLSLRTNE Q⁶⁷-M¹¹⁶ 13QINDIFQKLNIFDQSFYDLSLRTNETKEEEKELR peptide RTTSTLQVKNEEVKNM I⁹²-L¹⁴¹ 14IKEEEKELRRTTSTLQVKNEEVKNMSVELNSKLESLLEE peptide KTALQHKVRAL S¹¹⁷-S¹⁶⁶ 15SVELNSKLESLLEEKTALQHKVRALEEQLTNLIL peptide SPAGAQEHPEVTSLKS E¹⁴²-Q¹⁹¹ 16EEQLTNLILSPAGAQEHPEVTSLKSFVEQQDNSIRELLQ peptide SVEEQYKQLSQ F¹⁶⁵-L²¹⁶ 17FVEQQDNSIRELLQSVEEQYKQLSQQHMQIKEIE peptide KQLRKTGIQEPSENSL Q¹⁹²-D²⁴¹ 18QHMQIKEIEKQLRKTGIQEPSENSLSSKSRAPRTTPPLQ peptide LNETENTEQDD 4.7.1 heavy19 MEWSWIFLFL LSGTAGVHSE VQLQQSGPEL VKPGASVKMS chain variable CKASGYTFTSYVMHWVKQKP GQGLEWIGYF NPYNDGTKYN region EKFKGKATLT SDKSSSTAYM ELSSLTSEDSAVYYCAREGD YYGYFDYWGQ GTTLTVSSA 4.7.1 heavy 20 E VQLQQSGPEL VKPGASVKMSCKASGYTFTS chain variable YVMHWVKQKP GQGLEWIGYF NPYNDGTKYN EKFKGKATLTregion without SDKSSSTAYM ELSSLTSEDS AVYYCAREGD YYGYFDYWGQ signalpeptide GTTLTVSSA 4.8.3 heavy 21 MEWSWIFLFL LSGTAGVHSE VQLQQSGPELVKPGASVKMS chain variable CKASGYTFIS CVMHWVKQKP GQGLEWIGYI NPYNDGTKYNregion EKFKGKATLT SDKSSSTAYM ELSSLTSEDS AVYYCAREGD YYGYFDYWGQ GTTLTVSSA4.8.3 heavy 22 E VQLQQSGPEL VKPGASVKMS CKASGYTFIS chain variableCVMHWVKQKP GQGLEWIGYI NPYNDGTKYN EKFKGKATLT region without SDKSSSTAYMELSSLTSEDS AVYYCAREGD YYGYFDYWGQ signal peptide GTTLTVSSA 4.9.1 heavy 23MEWSWIFLFL LSGTAGVHSE VQLQQSGPEL VKPGASVKMS chain variable CKASGYTFTSYVMHWVKQKP GQGLEWIGYI NPYNDGTKYN region ENFKGKATLT SDKSSSTAYM EFSSLTSEDSAVYYCAREGD YYGYFDYWGQ GTTLTVSSA 4.9.1 heavy 24 E VQLQQSGPEL VKPGASVKMSCKASGYTFTS chain variable YVMHWVKQKP GQGLEWIGYI NPYNDGTKYN ENFKGKATLTregion without SDKSSSTAYM EFSSLTSEDS AVYYCAREGD YYGYFDYWGQ signalpeptide GTTLTVSSA heavy chain 25 MEWSWIFLFL LSGTAGVHSE VQLQQSGPELVKPGASVKMS variable re- CKASGYTFTS YVMHWVKQKP GQGLEWIGYI NPYNDGTKYN gionconsensus EKFKGKATLT SDKSSSTAYM ELSSLTSEDS AVYYCAREGD YYGYFDYWGQGTTLTVSSA heavy chain 26 E VQLQQSGPEL VKPGASVKMS CKASGYTFTS variable re-YVMHWVKQKP GQGLEWIGYI NPYNDGTKYN EKFKGKATLT gion consensus SDKSSSTAYMELSSLTSEDS AVYYCAREGD YYGYFDYWGQ without signal GTTLTVSSA peptide 4.7.1light 27 MSSAQFLGLL LLCFQGTRCD IQMTQTTSSL SASLGDRVTI chain variableSCRASQDISN FLNWYQQKPD GTVKLLIYYT SRLHSGVPSR region FSGSGSGTDY SLTISNLEQEDIATYFCQQG NTLPPTFGGG TKLEIKR 4.7.1 light 28 D IQMTQTTSSL SASLGDRVTISCRASQDISN chain variable FLNWYQQKPD GTVKLLIYYT SRLHSGVPSR FSGSGSGTDYregion without SLTISNLEQE DIATYFCQQG NTLPPTFGGG TKLEIKR signal peptide4.8.3 light 29 MSSAQFLGLL LLCFQGIRCE IQMTQTTSSL SASLGDRVTI chainvariable SCWASQDINN YLNWYQQKPD GTVKLLIYYT SRLHSGVPSR region FSGSGSGTDYSLTISNLKQE DIATYFCQQG NTLPPTFGGG TKLEIKR 4.8.3 light 30 E IQMTQTTSSLSASLGDRVTI SCWASQDINN chain variable YLNWYQQKPD GTVKLLIYYT SRLHSGVPSRFSGSGSGTDY region without SLTISNLKQE DIATYFCQQG NTLPPTFGGG TKLEIKRsignal peptide 4.9.1 light 31 MSSAQFLGLL LLCFQGARCD IQMTQTTSSLSASLGDRVTI chain variable SCRASQDIRN YLNWYQQKPD GTVKLLIYYT SRLHSGVPSRregion FSGSGSGTDY SLTISNLEQE DIATYFCQQG NTLPPTFGGG TKLEIKR 4.9.1 light32 D IQMTQTTSSL SASLGDRVTI SCRASQDIRN chain variable YLNWYQQKPDGTVKLLIYYT SRLHSGVPSR FSGSGSGTDY region without SLTISNLEQE DIATYFCQQGNTLPPTFGGG TKLEIKR signal peptide light chain 33 MSSAQFLGLL LLCFQGXRCDIQMTQTTSSL SASLGDRVTI variable re- SCRASQDIXN YLNWYQQKPD GTVKLLTYYTSRLHSGVPSR gion consensus FSGSGSGTDY SLTISNLEQE DIATYFCQQG NTLPPTFGGGTKLEIKR light chain 34 D IQMTQTTSSL SASLGDRVTI SCRASQDIXN variable re-YLNWYQQKPD GTVKLLIYYT SRLHSGVPSR FSGSGSGTDY gion consensus SLTISNLEQEDIATYFCQQG NTLPPTFGGG TKLEIKR without signal peptide 4.7.1 heavy 35GYTFTSYVMH chain CDR1 4.7.1 heavy 36 YFNPYNDGTKYNEKFKG chain CDR2 4.7.1heavy 37 EGDYYGYFDY chain CDR3 4.8.3 heavy 38 GYTFISCVMH chain CDR14.8.3 heavy 39 YINPYNDGTKYNEKFKG chain CDR2 4.8.3 heavy 40 EGDYYGYFDYchain CDR3 4.9.1 heavy 41 GYTFTSYVMH chain CDR1 4.9.1 heavy 42YINPYNDGTKYNENFKG chain CDR2 4.9.1 heavy 43 EGDYYGYFDY chain CDR3 4.7.1light 44 RASQDISNFLN chain CDR1 4.7.1 light 45 YTSRLHS chain CDR2 4.7.1light 46 QQGNTLPPT chain CDR3 4.8.3 light 47 WASQDINNYLN chain CDR14.8.3 light 48 YTSRLHS chain CDR2 4.8.3 light 49 QQGNTLPPT chain CDR34.9.1 light 50 RASQDIRNYLN chain CDR1 4.9.1 light 51 YTSRLHS chain CDR24.9.1 light 52 QQGNTLPPT chain CDR3 heavy chain 53 GYTFTSYVMH CDR1consensus heavy chain 54 YINPYNDGTKYNEKFKG CDR2 consensus heavy chain 55EGDYYGYFDY CDR3 consensus light chain 56 RASQDIXNYLN CDR1 consensuslight chain 57 YTSRLHS CDR2 consensus light chain 58 QQGNTLPPT CDR3consensus mouse ANGPTL3 59 SRVD PDLSSFDSAP SEPKSRFAML DDVKILANGLS¹⁷-D²⁴⁰ LQLGHGLKDF VHKTKGQIND IFQKLNIFDQ SFYDLSLRTN EIKEEEKELRRTTSTLQVKN EEVKNMSVEL NSKLESLLEE KTALQHKVRA LEEQLTNLIL SPAGAQEHPEVTSLKSFVEQ QDNSIRELLQ SVEEQYKQLS QQHMQIKEIE KQLRKTGIQE PSENSLSSKSRAPRTTPPLQ LNETENTEQD human ANGPTL3 60 D QDNSSFDSLS PEPKSRFAMLDDVKILANGL S¹⁷-D²⁴⁰ LQLGHGLKDF VHKTKGQIND IFQKLNIFDQ SFYDLSLQTSEIKEEEKELR RTTYKLQVKN EEVKNNSLEL NSKLESLLEE KILLQQKVKY LEEQLTNLIQNQPETPEHPE VTSLKTFVEK QDNSIKDLLQ TVEDQYKQLN QQHSQIKEIE NQLRRTSIQEPTEISLSSKP RAPRTTPFLQ LNEIRNVKHD GIP mouse ANGPTL3 61 DVKILANGLLQLGHGLKDF VHKTKGQIND IFQKLNIFDQ D⁴²-M¹¹⁶ SFYDLSLRTN EIKEEEKELRRTTSTLQVKN EEVKNM SP2-KLH 62 EPKSRFAMLDDVKILANGLLQLGHGLC 5.35 heavy 63MGRLTSSFLL LIVPAYVLSQ VTLKESGPGI LHPSQTLTLT chain variable CSFSGFSLNTFGLAVGWIRQ PSGKGLEWLG HIWWDDHKYY region NGVLKSRLTI SKDSSKKQVF LRIANVDTADTARYYCARLE TGTGFAYWGQ GTLVTVSAA 5.35 heavy 64 Q VTLKESGPGI LHPSQTLTLTCSFSGFSLNT chain variable FGLAVGWIRQ PSGKGLEWLG HIWWDDHKYY NGVLKSRLTIregion without SKDSSKKQVF LRIANVDTAD TARYYCARLE TGTGFAYWGQ signalpeptide GTLVTVSAA 5.50 heavy 65 MGWSWIFLFL LSGTAGVLSE VQLQQSGPELVKPGASVKIS chain variable CKASCFTFTD YYMNWVKQSH GESLEWIGDI NPNNGGTIYNregion QKFRGKATLT VDKSSSTAYM ELRSLTSEDS AVYYCVRLPW YFDVWGTGTT VTVSSA5.50 heavy 66 E VQLQQSGPEL VKPGASVKIS CKASGFTFTD chain variableYYMNWVKQSH GESLEWIGDI NPNNGGTIYN QKFRGKATLT region without VDKSSSTAYMELRSLTSEDS AVYYCVRLPW YFDVWGTGTT signal peptide VTVSSA 5.35 light 67MRCLAEFLGL LVLWIPGAIG DIVLTQSTPS VPVTPGESVS chain variable ISCRSSKSLLDSNGITYLYW FLQRPGQSPQ LLIYRMSKLA region SGVPDRFSGS GSETAFTLRI SRVEAEDVGVYYCMQPLEYP FTFGAGTKLE LNG 5.35 light 68 DIVLTQSTPS VPVTPGESVS ISCRSSKSLLDSNGITYLYW chain variable FLQRPGQSPQ LLIYRMSKLA SGVPDRFSGS GSETAFTLRIregion without SRVEAEDVGV YYCMQPLEYP FTFGAGTKLE LNG signal peptide 5.50light 69 MKLPVRLLVL MFWIPASSSD VLMTQTPLSL PVSLGDQASI chain variableSCRSSQSILH SNGNTYLEWF LQKPGQSPKL LIYKVSNRFS region GVPDRFSGSG SGTDFTLKISRVEAEDLGVY YCFQGSHVPY TFGGGTKLEI KR 5.50 light 70 D VLMTQTPLSLPVSLGDQASI SCRSSQSILH chain variable SNGNTYLEWF LQKPGQSPKL LIYKVSNRFSGVPDRFSGSG region without SGTDFTLKIS RVEAEDLGVY YCFQGSHVPY TFGGGTKLEIsignal peptide KR 5.35 heavy 71 GFSLNTFGLAVG chain CDR1 5.35 heavy 72HIWWDDHKYYNGVLKS chain CDR2 5.35 heavy 73 LETGTGFAY chain CDR3 5.50heavy 74 GFTFTDYYMN chain CDR1 5.50 heavy 75 DINPNNGGTIYNQKFRG chainCDR2 5.50 heavy 76 LPWYFDV chain CDR3 5.35 light 77 RSSKSLLDSNGITYLYchain CDR1 5.35 light 78 RMSKLAS chain CDR2 5.35 light 79 MQPLEYPFTchain CDR3 5.50 light 80 RSSQSILHSNGNTYLE chain CDR1 5.50 light 81KVSNRFS chain CDR2 5.50 light 82 FQGSHVPYT chain CDR3 mutant 1 83APKSRFAMLDDVKILANGLLQLGHGL mutant 2 84 EAKSRFAMLDDVKILANGLLQLGHGL mutant3 85 EPASRFAMLDDVKILANGLLQLGHGL mutant 4 86 EPKARFAMLDDVKILANGLLQLGHGLmutant 5 87 EPKSAFAMLDDVKILANGLLQLGHGL mutant 6 88EPKSRAAMLDDVKILANGLLQLGHGL mutant 8 89 EPKSRFAALDDVKILANGLLQLGHGL mutant9 90 EPKSRFAMADDVKILANGLLQLGHGL mutant 10 91 EPKSRFAMLADVKILANGLLQLGHGLmutant 11 92 EPKSRFAMLDAVKILANGLLQLGHGL mutant 12 93EPKSRFAMLDDAKILANGLLQLGHGL mutant 13 94 EPKSRFAMLDDVAILANGLLQLGHGLmutant 14 95 EPKSRFAMLDDVKALANGLLQLGHGL mutant 15 96EPKSRFAMLDDVKIAANGLLQLGHGL mutant 17 97 EPKSRFAMLDDVKILAAGLLQLGHGLmutant 18 98 EPKSRFAMLDDVKILANALLQLGHGL mutant 19 99EPKSRFAMLDDVKILANGALQLGHGL mutant 20 100 EPKSRFAMLDDVKILANGLAQLGHGLmutant 21 101 EPKSRFAMLDDVKILANGLLALGHGL mutant 22 102EPKSRFAMLDDVKILANGLLQAGHGL mutant 23 103 EPKSRFAMLDDVKILANGLLQLAHGLmutant 24 104 EPKSRFAMLDDVKILANGLLQLGAGL mutant 25 105EPKSRFAMLDDVKILANGLLQLGHAL mutant 26 106 EPKSRFAMLDDVKILANGLLQLGHGAmouse ANGPTL4 107 mrcaptagaa lvlcaatagl lsaqgrpaqp epprfaswde (AccessionNo. mnllahgllq lghglrehve rtrgqlgale rrmaacgnac NP_065606) qgpkgkdapfkdsedrvpeg qtpetlqslq tqlkaqnski qqlfqkvaqq qrylskqnlr iqnlqsqidllapthldngv dktsrgkkls kmtqliglts nathlhrpar dcqelfqege rhsglfqiqplgsppflvnc emtsdggwtv iqrrlngsvd fnqsweaykd gfgdpqgefw lglekmhsitgdrgsqlavq lqdwdgnakl lqfpihlgge dtayslqlte ptanelgatn vspnglslpfstwdqdhdlr gdlncaksls ggwwfgtcsh snlngqyfhs iprqrqerkk gifwktwkgryyplqattll iqpmeataas human ANGPTL4 108 msgaptagaa lmlcaatavl lsaqggpvqsksprfaswde (Accession No. mnvlahgllq lgqglrehae rtrsqlsale rrlsacgsacNP_647475) qgtegstdlp lapesrvdpe vlhslqtqlk aqnsriqqlf hkvaqqqrhlekqhlriqhl qsqfglldhk hldhevakpa rrkrlpemaq pvdpahnvsr lhrlprdcqelfqvgerqsg lfeiqpqgsp pflvnckmts dggwtviqrr hdgsvdfnrp weaykagfgdphgefwlgle kvhsitgdrn srlavqlrdw dgnaellqfs vhlggedtay slqltapvagqlgattvpps glsvpfstwd qdhdlrrdkn cakslsggww fgtcshsnln gqyfrsipqqrqklkkgifw ktwrgryypl qattmliqpm aaeaas mouse ANGPTL4 109 QPEPPRFASWDEMNLLAHGL LQLGHGL SP1 region human ANGPTL4 110 SKSPRFASWD EMNVLAHGLLQLGQGL SP1 region

1. A monoclonal antibody that binds to ANGPTL3 and neutralizes at leastone activity of ANGPTL3.
 2. The monoclonal antibody of claim 1, whereinthe monoclonal antibody is a mouse monoclonal antibody.
 3. Themonoclonal antibody of claim 1, wherein the monoclonal antibody is ahumanized monoclonal antibody.
 4. The monoclonal antibody of claim 1,wherein the monoclonal antibody is a human monoclonal antibody.
 5. Themonoclonal antibody of claim 1, wherein the monoclonal antibodydecreases the level of at least one serum lipid in vivo.
 6. Themonoclonal antibody of claim 1, wherein the monoclonal antibody binds toan epitope of ANGPTL3 having the amino acid sequence of SEQ ID NO: 59.7. The monoclonal antibody of claim 1, wherein the monoclonal antibodybinds to an epitope of ANGPTL3 having the amino acid sequence of SEQ IDNO:
 60. 8. The monoclonal antibody of claim 1, wherein the monoclonalantibody binds to an epitope of ANGPTL3 having the amino acid sequenceof SEQ ID NO:
 9. 9. The monoclonal antibody of claim 1, wherein themonoclonal antibody binds to an epitope of ANGPTL3 having the amino acidsequence of SEQ ID NO:
 10. 10. The monoclonal antibody of claim 1,wherein the monoclonal antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 20 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:
 28. 11.The monoclonal antibody of claim 1, wherein the monoclonal antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 22 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:
 30. 12. The monoclonal antibody ofclaim 1, wherein the monoclonal antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 24 anda light chain variable region comprising the amino acid sequence of SEQID NO:
 32. 13. The monoclonal antibody of claim 1, wherein themonoclonal antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 64 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:
 68. 14. Themonoclonal antibody of claim 1, wherein the monoclonal antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 66 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:
 70. 15. The monoclonal antibody ofclaim 1, wherein the monoclonal antibody specifically binds to the sameepitope as antibody 4.7.1.
 16. The monoclonal antibody of claim 1,wherein the monoclonal antibody specifically binds to the same epitopeas antibody 4.8.3.
 17. The monoclonal antibody of claim 1, wherein themonoclonal antibody specifically binds to the same epitope as antibody4.9.1.
 18. The monoclonal antibody of claim 1, wherein the monoclonalantibody specifically binds to the same epitope as antibody 1.315.1. 19.The monoclonal antibody of claim 1, wherein the monoclonal antibodyspecifically binds to the same epitope as antibody 5.35.
 20. Themonoclonal antibody of claim 1, wherein the monoclonal antibodyspecifically binds to the same epitope as antibody 5.50. 21.-64.(canceled)
 65. A monoclonal antibody that binds to ANGPTL3 andneutralizes at least one activity of ANGPTL3, wherein the antibody bindsto a peptide having the amino acid sequence of SEQ ID NO: 9 with a K_(D)of less than 50 nM.
 66. The monoclonal antibody of claim 65, wherein theantibody binds to a peptide having the amino acid sequence of SEQ ID NO:9 with a K_(D) of less than 30 nM.
 67. The monoclonal antibody of claim65, wherein the antibody binds to a peptide having the amino acidsequence of SEQ ID NO: 9 with a K_(D) of less than 10 nM.
 68. Themonoclonal antibody of claim 65, wherein the antibody binds to a peptidehaving the amino acid sequence of SEQ ID NO: 9 with a K_(D) of less than5 nM. 69.-77. (canceled)